Sadee W, Hoeg E, Lucas J and Wang D Genetic Variations in Human G Protein-Coupled Receptors: Implications for Drug Therapy AAPS PharmSci 2001; 3 (3) article 22 (https://www.pharmsci.org/scientificjournals/pharmsci/journal/01_22.html).

Genetic Variations in Human G Protein-Coupled Receptors: Implications for Drug Therapy

Submitted: March 4, 2001; Accepted: June 30, 2001; Published: July 26, 2001

Wolfgang Sadee¹, Elen Hoeg¹, Julie Lucas¹ and Danxin Wang¹

¹Departments of Biopharmaceutical Sciences and Pharmaceutical Chemistry, University of California San Francisco, San Francisco CA 94143-0446

Abstract

Numerous genes encode G protein-coupled receptors (GPCRs)-a main molecular target for drug therapy. Estimates indicate that the human genome contains approximately 600 GPCR genes. This article addresses therapeutic implications of sequence variations in GPCR genes. A number of inactivating and activating receptor mutations have been shown to cause a variety of (mostly rare) genetic disorders. However, pharmacogenetic and pharmacogenomic studies on GPCRs are scarce, and therapeutic relevance of variant receptor alleles often remains unclear. Confounding factors in assessing the therapeutic relevance of variant GPCR alleles include 1) interaction of a single drug with multiple closely related receptors, 2) poorly defined binding pockets that can accommodate drug ligands in different orientations or at alternative receptor domains, 3) possibility of multiple receptor conformations with distinct functions, and 4) multiple signaling pathways engaged by a single receptor. For example, antischizophrenic drugs bind to numerous receptors, several of which might be relevant to therapeutic outcome. Without knowing accurately what role a given receptor subtype plays in clinical outcome and how a sequence variation affects drug-induced signal transduction, we cannot predict the therapeutic relevance of a receptor variant. Genome-wide association studies with single nucleotide polymorphisms could identify critical target receptors for disease susceptibility and drug efficacy or toxicity.

Introduction

Sequence variations of the human genome.

This article provides an overview of the large superfamily of G protein-coupled receptors (GPCRs) and its variant alleles in the human population known to affect receptor function (Table 1 ) ^1-132 . Sequencing of the human genome has introduced a flood of new information on the projected approximately 35 000 genes ^133,134 ; however, the primary sequence is but a first step in understanding genomic organization, protein functions, communication networks, and cellular structure. Furthermore, the presence of sequence variations introduces a near-infinite variability in the genetic makeup of individuals. This is suspected to play a main role in disease susceptibility and variable response to drug therapy. The latter is the subject of pharmacogenetics-pharmacogenomics-with pharmacogenomics focusing on the entire genome or using genomic techniques to design and develop new drugs and guide therapy.

Polymorphisms refer to sequence variations with an allele frequency of greater than or equal to 1%; however, mutant alleles responsible for sporadic single-gene Mendelian diseases are often much less frequent. The exchange of a single nucleotide, commonly referred to as single nucleotide polymorphisms (SNPs), accounts for approximately 80% of all sequence variants. Current estimates of SNP frequency are 1:1200 ¹³³ , but this is clearly a function of coverage for genome sequencing (4- to 8-fold coverage). With increasing coverage (ie, more overlapping sequences analyzed from different individuals), SNP abundance will increase further.

Given a gene encoding a GPCR of an average length (1000-1500 base pair [bp] coding region), we would expect to find an average of 1 relatively common SNP and several more SNPs with a frequency of more than 1%. Table 1 lists a selection of known sequence variants identified in human GPCR genes ^1-132 . Because a majority of the listed SNPs have a relatively low allele frequency, an individual likely will not harbor a sequence variation at all in a GPCR gene. Even though this review focuses on sequence differences among individuals, we should recognize the extraordinary degree of conservation of a molecule as brittle as DNA across the human population. Sequence variants might accumulate in a population if they convey a selective advantage to the individual carrying the allele, but there is no such evidence for GPCR alleles. Chemokine receptors serving as coacceptors for the acquired immunodeficiency syndrome (AIDS) virus for penetration into cells could represent an exception (Table 1 ) because inactivating mutations appear to convey resistance to human immunodeficiency virus (HIV) infection ^109,110 . However, AIDS has entered the human population only recently, precluding positive selection of inherited traits, which requires numerous generations.

Most polymorphisms in a GPCR gene are unlikely to affect receptor function, either because they occur in noncoding regions of the mature mRNA or in introns. Alternatively, SNPs occurring in coding regions can be silent (synonymous, no change in protein sequence) or occur in a region that can accommodate amino acid substitutions without functional consequences. Yet, we have only a partial understanding of all functional receptor domains that interact with ligands and numerous other proteins mediating receptor function. Polymorphisms in promoter regions or at splice junctions can have profound effects on the abundance of the encoded protein. A growing number of recognized polymorphisms in GPCR promoter regions suggests the importance of overall receptor expression of interindividual variability, but examples are still scarce. Here, we review general GPCR structure and function to facilitate a better understanding of how sequence variants might affect receptor signaling and drug interactions.

Structure and function of GPCRs

GPCRs comprise a large class of membrane proteins that are encoded by approximately 600 human genes with broadly diverse functions ¹³⁵ . Venter et al ¹³³ predict the presence of 614 GPCRs, a number that requires further verification but is probably close to the true number of genes in this class. Ligands are extremely diverse and include hormones and neurotransmitters and neuromodulators such as biogenic amines, amino acids, peptides, glycoproteins, prostanoids, phospholipids, nucleosides and nucleotides, light-retinal, olfactants, and Ca²⁺ .

To understand the possible effects of sequence variations, it is necessary to analyze the molecular architecture of GPCRs. Moreover, we need to address the questions of whether and how GPCRs are related to each other in evolution. This might permit the prediction of functionally relevant domains where sequence variations are most likely to alter receptor function. Lastly, the extraordinary multiplicity of GPCRs represents a critical-and possibly a limiting-factor in our ability to predict the physiological effects of a mutation in a single receptor because of redundancy in signaling networks.

GPCR structure.

Biochemical and biophysical investigations show that GPCRs share a common overall structure characterized by 7 tandemly arranged transmembrane domains (TMDs) (Figure 1 ; for more snake-like views of GPCRs, see Table 1 , link #3). Because of constraints imposed on their structures by their localization in the cellular membrane, TMDs can be identified by hydropathy analysis and are predicted to be a-helical structures, usually consisting of 20 to 24 amino acids each. These structures are linked through loops that intrude either into the extracellular space (e1-3) or the cytosol (i1-3) and are flanked by an extracellular N-terminal and an intracellular C-terminal tail. Whereas the transmembrane domains are highly conserved among closely related GPCRs, the loops are more variable in sequence and length, and the C- and N-terminal tails represent the most diverse elements.

A number of GPCR genes exist as a single exon, suggesting that gene duplications have involved a mechanism of retroposition. However, many GPCR genes are multiexonic; therefore, we must expect the existence of splice variants with distinct functions, as has been demonstrated for the prostaglandin EP3 receptor subtype. Alternative splicing of EP3 yields at least 4 isoforms that differ in their C-terminus and couple to different G proteins and second messengers¹³⁶ . Many more splice variants can be expected that have yet to be studied (for a review, see ¹³⁷ ).

GPCR ancestry.

Despite compelling similarity in GPCRs' overall structure, the lack of statistically significant sequence similarity among several GPCR families raises the question of whether all GPCRs arose through common ancestry. Thus, vasoactive intestinal peptide, secretin, and metabotropic glutamate receptors show little sequence similarity to other peptide and biogenic amine receptors. In an attempt to understand evolutionary relationships, we have classified the sequences of approximately 1700 GPCRs and unrelated membrane proteins into clusters on the basis of sequence similarities ¹³⁵ . Taking advantage of the dramatically increased number of cloned GPCRs from many species, this approach resulted in significant alignments between distant GPCR families, including receptors for the biogenic amine/peptide, vasoactive intestinal peptide/secretin, cyclic adenosine monophosphate (cAMP), STE3/MAP3 fungal pheromones, latrophilin, developmental receptors frizzled and smoothened, as well as the more distant metabotropic glutamate receptors. This study provides a refined view of GPCR ancestry, displays conserved sequence motifs for each receptor cluster, and serves as a reference database with hyperlinks to other sources ¹³⁵ . Nevertheless, the numerous functionally diverse GPCR families often show marginal sequence similarities; therefore, care has to be taken when inferring structure-function relationships by comparing GPCRs from different families. Specifically, we cannot readily extrapolate the effect of sequence variations on structure and function of 1 receptor cluster to another.

GPCR coupling to G proteins and other signaling pathways.

As implied by the name, GPCRs are thought to couple to heterotrimeric G proteins composed of a, b and g subunits. However, direct proof for G protein coupling remains elusive for the majority of the approximately 600 human GPCRs. G proteins also display considerable heterogeneity, with a predicted number of 27 different a, 5 b, and 13 g subunits¹³³ . Upon receptor activation, GDP dissociates from the a subunit, and GTP binds to and activates the G protein. This leads to dissociation of Ga and Gbg, each capable of triggering multiple downstream events. Main pathways include the regulation of adenylyl cyclases and cAMP phosphodiesterases, phospholipase C pathways, and regulation of ion channel activity. Taking advantage of the inherent GTPase activity of the Ga subunit, the activation process is reversed by production of Ga/GDP and its reassociation with Gbg. Signal transduction is made more complex by the ability of a single receptor to engage multiple Ga proteins. Moreover, receptor recognition and signaling pathways are also determined by b andg subunits¹³⁷ . Thus, overall effects of receptor activation can have opposite results in different tissues, as shown for the m4 muscarinic receptor-depending on which G proteins are expressed and which signaling molecules are present¹³⁸ . Main sites of contact between receptor and G proteins include the third intracellular loop (i3), but i1, i2, and the C-terminus have also been reported to contribute G protein coupling^139-141 . Therefore, the residues critical for coupling need to be determined individually for each receptor subgroup.

In addition to G proteins, GPCRs are known to interact with many other proteins, some of which may also serve signaling functions¹⁴² . Receptor-associated proteins include arrestins, protein kinases and phosphatases, PDZ-domain binding proteins (if a C-terminal PDZ consensus sequence is present), and various modifying enzymes; for example, those introducing palmityl residues into the C-terminus. Each of these proteins modulates receptor functions at distinct domains that are possible targets for polymorphic effects in human GPCR signaling.

We have recently determined that the opioid receptor domain involved in G protein coupling (i3 loop) also interacts directly with calmodulin (Figure 2 ) ^143,144 . Upon receptor activation, calmodulin is displaced from the receptor, thereby allowing G protein coupling to proceed while calmodulin itself appears to serve as a novel receptor messenger¹⁴⁵ . Hence, reported sequence variants of µ-opioid receptor (MOR) in its i3 loop could affect either G protein coupling, calmodulin binding, or both (see the following for polymorphic effects). It remains to be seen whether this is a general phenomenon for GPCRs.

GPCR binding pockets

The astounding diversity of receptor ligands begs the question of where the binding site resides and how it is structured. It is inconceivable that Ca²⁺ , acetylcholine, glutamate, bradykinin, prostaglandins, and the large polypeptide follicle-stimulating hormone all bind to the same site. Indeed, for each of these ligands, distinct binding sites appear to exist, either embedded within the pocket formed by the 7-TMD bundle within the membrane (biogenic amines), at pockets formed by the extracellular loops (peptides), or in the N-terminus (glutamate, Ca²⁺ , glycoprotein hormones)¹⁴⁶ . The latter may consist of an evolutionarily distinct protein module. For example, Ca²⁺ , glutamate, g acid (GABA), and certain pheromones bind to a large N-terminal protein module related in evolution to the periplasmic binding proteins of gram-negative bacteria¹⁴⁷ (Figure 3 ). On the other hand, the thrombin receptor family represents a special case where the protease activity of the ligand thrombin cleaves a portion of the N-terminus. The newly generated N-terminus then serves as a tethered ligand for the receptor, rendering it constitutively active until degraded¹⁴⁸ .

These findings indicate that there is no parsimonious receptor-binding pocket as expected for the catalytic site of enzymes. Rather, GPCRs appear to be activated by ligand binding to many different sites of the protein. At the opioid receptors, peptide endorphins bind primarily to the extracellular loops, whereas opioid alkaloids dock deep into the 7-TMD core¹⁴⁹ . Thus, a single receptor can be activated by various ligands binding to several distinct, often overlapping sites. Even within the same binding pocket, there is no invariant set of amino acid residues contributing to ligand binding. Studying a series of opioid ligands, Befort et al¹⁵⁰ have found that different residues appear to participate in the binding pocket of d-opioidreceptor (DOR) even for closely related opioid compounds. This suggests that ligands can bind into the receptor pocket with different orientations, which may be affected by very small changes in chemical structure, including stereoisomers.

In summary, sequence variations in the receptor protein can affect ligand binding or the structural integrity of the receptor, indirectly changing ligand binding. Alternatively, mutations can alter G protein coupling or cellular trafficking such that the receptor is no longer expressed at the cell surface ¹⁴⁹ .

Spontaneous GPCR signaling

GPCRs tend to show spontaneous, basal signaling activity in the absence of agonists (also referred to as constitutive activity)^151-153 . Constitutive activity of wild-type b2-adrenergic¹⁵⁴ , serotonin^155,156 , bradykinin¹⁵⁷ , d-opioid¹⁵⁸ , and muscarinic¹⁵⁹ receptors has been reported. Constitutive activity of MOR¹⁶⁰ , and in particular its up-regulation following chronic treatment with opiates, has been hypothesized to account for part of the regulatory mechanism underlying narcotic tolerance and dependence^{144, 161} .

By altering the primary structure of GPCRs with site-directed mutagenesis, a number of investigators have found that exchange of single amino acid residues can lead to constitutive receptor activation¹⁶² . Surprisingly, activating point mutations do not map to any specific area but are distributed throughout the receptor protein^{146, 163} . This parallels the finding of different ligand binding activation domains. A possible conclusion derived from these studies is that GPCRs generally exist in a constrained inactive conformation that requires some trigger for activation by folding into a more relaxed structure. Indeed, a considerable number of human polymorphisms enhance signaling (gain of function) or even activate the receptor constitutively, causing serious genetic disorders (Table 1 ). Such mutant alleles are usually dominant and present opportunities for therapeutic intervention.

Basal signaling activity is frequently observed in cell lines in which receptors are overexpressed¹⁵⁴ , but in some cases, basal activity is independent of receptor density^164,165 . This suggests that basal signaling represents an inherent physiological characteristic of the receptor. By definition, antagonists block agonist-mediated activation, but they can have distinct effects at basally active receptors. Those agonists suppressing basal signaling activity of the receptor are referred to as inverse agonists, or antagonists with negative intrinsic activity, whereas neutral antagonists or antagonists with no intrinsic activity do not affect basal signaling^{153, 165} . Inverse agonists and neutral antagonists have been identified for a number of GPCRs. This becomes relevant for the treatment of inherited disorders caused by activating GPCR mutations, but inverse agonists have yet to be used clinically.

Lastly, activating mutations, or spontaneously active receptors, can serve as mitogens or oncogenes. This was first discovered by site-directed mutagenesis, rendering GPCRs spontaneously active. For example the a1B-adrenergic receptor becomes mitogenic upon introduction of activating mutations in the C-terminal portion of the third intracellular (i3) loop¹⁶⁶ . The physiological and pharmacological relevance of basal receptor signaling and the role of naturally occurring variant alleles will be discussed in more detail later (see also Table 1 ).

Multiple receptor conformations with distinct functions

Another possible explanation for unpredictable effects of receptor mutations on ligand binding is that GPCRs are flexible structures and may accommodate ligands in various ways. Indeed, GPCRs have been suspected to exist in multiple conformations. Moreover, recent evidence supports the view that discrete conformational states of GPCRs trigger distinct signaling pathways. For example, octopamine and tyramine each stimulate a separate signaling pathway at their common receptor in Drosophila ¹⁶⁷ . An activating mutation of the a1B-adrenergic receptor selectively stimulates only 1 of 2 a1B signaling pathways examined¹⁶⁸ . Similarly, structurally distinct ligands differentially activate Gi and Go coupling of cannabinoid receptors¹⁶⁹ . Different MOR agonists vary dramatically in their ability to induce receptor internalization^170,171 . The opioid peptide DAMGO and etorphine, but not morphine, were shown to cause receptor internalization, even though all 3 strongly stimulate G protein coupling. This distinguishes receptor forms active in coupling and internalization. Using various ligands and site-directed mutagenesis, Thomas et al¹⁷² have demonstrated the existence of multiple receptor conformations of the angiotensin II receptor, each supporting distinct functions: G protein coupling, internalization, and receptor phosphorylation. Lastly, numerous ligand-binding studies have revealed the existence of multiple receptor conformations^{156, 173,174} . Clearly, this makes it difficult to predict which residues of a receptor will prove relevant for binding a given ligand, impeding rapid progress in receptor pharmacogenetics.

GPCR aggregation

Recent evidence suggests that essential molecules of GPCR signaling pathways are held in close proximity of each other in microdomains such as caveolae and are not freely floating or dependent on random collision to interact¹⁷⁵ . Therefore, access of ligands to receptor microdomains may differ between polar and lipophilic ligands. On the other hand, multiple receptor conformations and complexes might exist that are associated with different signaling pathways via the proteins contained within the complex. Target size analysis of GPCRs in the plasma membrane has revealed the existence of very large GPCR complexes exceeding 1 million d, which partially break up on agonist stimulation¹⁷⁶ . Receptor aggregation as a main organizing principle could lead to oligomeric receptors and functional complexes. Specifically, the DOR was shown to dimerize with itself and with the k opioid receptor (KOR)¹⁷⁷ , whereas the MOR forms oligomers with itself and DOR¹⁷⁸ . Homo- and hetero-oligomerization have been shown to affect the functional properties of these and other GPCRs^179,180 . The presence of low- and high-agonist affinity sites has been associated with formation of a receptor-G protein complex¹⁷³ but may also be related to receptor oligomerization as suggested for the m2 muscarinic receptor¹⁷⁴ . Lastly, GPCRs may be in physical contact with ion channels, as shown for a dopamine D5-GABA-A channel¹⁸¹ . Some of the receptor domains responsible for aggregation have been described, often involving the intracellular C-terminus, but other domains are also likely to contribute. For example, the extracellular binding domains of metabotropic glutamate and GABA receptors are expected to dimerize in a fashion similar to that of periplasmic binding proteins¹⁴⁷ . Moreover, residues in the transmembrane segments may also support oligomerization. As a result, we expect numerous regions of GPCRs to interact with other proteins and promote aggregation in the membrane. Because most putative contact points are unknown, nonsynonymous sequence variants in any portion of the GPCR proteins must be analyzed for functional effect with all currently available assays to establish whether functional changes have occurred.

Receptor multiplicity and drug selectivity.

Through a variety of mechanisms, genes encoding GPCRs have duplicated and spread throughout eukaryotic genomes. Yeast, for example, contains several pheromone receptors and a glucose sensing GPCR, gpr1. The number of proposed GPCRs in the nematode, fruit fly, and human is 248, 146, and 616, respectively¹³³ . However, there are numerous additional chemo-attractant GPCR-like receptors in nematodes; classification of what constitutes a GPCR may not have been uniformly applied. Closely related genes (ie, those that have duplicated rather late in evolution) may locate next to each other on the same chromosome (tandem duplication, opsins, and olfactory receptors) or on separate chromosomes through translocations. Thus, at least 5 closely related human genes encode muscarinic cholinergic receptors, 5 encode dopamine receptors, and at least 15 encode serotonin receptors. Among these very closely related receptors, the TMDs are often most highly conserved. Because their binding pockets reside within the 7-TMD core, one can readily understand the difficulties in designing receptor subtype-specific drugs. Rather, most central nervous system-active drugs currently in clinical use bind to multiple drug receptors of the same subfamily and, furthermore, cross over to other receptor subfamilies. Chlorpromazine is one of the most promiscuous examples, binding to multiple dopamine, serotonin, and muscarinic acetylcholine receptors. Indeed, the spectrum of affinities to these receptors is thought to play a role in determining efficacy of antischizophrenic drugs, but it is exceedingly difficult to ascertain which receptor subtypes are critical.

Glutamate, GABA, serotonin, and acetylcholine are more commonly known as ligands for ion channels (ionotropic) rather than for GPCRs (metabotropic). Because of the considerably different structure of these ion channels, cross-reactivities at ionotropic receptors for drugs targeting GPCRs are less likely. However, for glutamate and GABA metabotropic and glutamate ionotropic receptors, binding sites share the same origin in evolution, namely the periplasmic binding proteins¹⁴⁷ . This module closes around the ligand-as in a firefly trap-thereby activating the ion channel or GPCR tethered to it (Figure 3 ). Because of this homology, the presence of cross-affinities between receptors and ion channels is conceivable.

Lack of receptor specificity presents a formidable challenge to pharmacogenetic-pharmacogenomic studies. Because schizophrenia is thought to have a multigenic origin, one might suspect that the spectrum of receptor subtype selectivities could determine clinical efficacy of a given drug in the individual patient. Genes involved in the etiology of schizophrenia are under intense investigation, providing the basis on which we may be able to rationally select the optimal drug regimen for individual patients. Likely, genes other than GPCRs will play key roles as well, such as the recently suspected RGS4 locus.

These examples illustrate the complexity of the GPCR signal transduction system. Each cell expresses countless GPCRs that trigger numerous interrelated signaling events. Loss of a functional receptor in a knockout experiment often has surprisingly little effect on overall physiology and behavior of the animal-as shown for the opioid receptors-either because the receptor does not display a basal tone in vivo or because other GPCRs compensate for the defect. However, drug effects can change profoundly. We must be most careful in interpreting sequence variants and their relevance to disease susceptibility and drug efficacy when taken together.

Sequence variations of GPCRs and associated diseases

In view of the large number of GPCRs in the human genome and their critical function in regulating cell behavior, a surprisingly small number of receptor variants have been linked to genetic diseases^{146, 163, 182,183} (or search the OMIM; Online Mendelian Inheritance in man) database for GPCRs:https://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?db=OMIM . Use the pull-down menu and select OMIM, or use link 2 provided in Table 1 ). Similarly, only a few of the sequence variations are known to alter drug effects. We will first summarize sequence variants associated with disease or specific phenotypes, without attempting to be comprehensive ( Table 1 ).

Impaired or enhanced agonist signaling efficacy.

Several inactivating sequence variants of peptide receptors have been associated with congenital disorders. For example, a point mutation causing truncation of the thyrotropin-stimulating hormone (TSH) receptor leads to Leydig's cell hypoplasia, and inactivating mutations of the adrenocorticotropic hormone (ACTH) receptor (MC2 receptor) are associated with familial glucocorticoid deficiency ⁷² . Some receptor variants display enhanced sensitivity to agonists, as reported for the angiotensin II type 1 receptor and the D72E variant of the TSH receptor. The latter mutation occurs in the large N-terminus, the binding site for glycoprotein hormone receptors, leading to toxic multinodular goiter ⁸⁶ .

V2 vasopressin receptor

A number of mutations in the gene encoding the V2 vasopressin receptor lead to functionally inactive receptor protein and are causative for nephrogenic diabetes insipidus (Figure 4 )^88,89 . V2 receptors recruit aquaporin-2 channels in the renal collecting ducts responsible for water retention. Thus, inactivating mutations of aquaporin-2 also result in nephrogenic diabetes insipidus¹⁸⁴ . This is a clear indication that receptor activity depends on intact signaling pathways with multiple components, each of which is subject to genetic variability.

The truncation mutation of the V2 vasopressin receptor provides a specific example, which suggests a possible therapeutic intervention. One of the more prevalent missense mutations inserts a termination codon leading to a receptor truncated within the i3 loop^88,89 . The N-terminal fragment consisting of TMDs 1-5 is nonfunctional as a GPCR. If one coexpresses the C-terminal fragment consisting only of the i3 loop, TMDs 6 and 7, and the C-tail, the 2 receptor fragments combine, traffic to the plasma membrane, and display at least partial receptor signaling activity⁸⁹ . This could provide an attractive strategy for gene therapy because the C-fragment per se would be inactive and functional receptor would be reconstituted only where the N-terminal fragment is expressed under the normal promoters of the V2 receptor gene.

Thromboxane A2 (TBXA2) receptor

The TBXA2 receptor performs an essential role in hemostasis by inducing platelet aggregation. An R60L amino acid substitution in the first cytoplasmic loop of the TBXA2 receptor causes a dominantly inherited bleeding disorder characterized by defective platelet response to TBXA2^120,121 . The mutant receptor showed decreased agonist-induced second messenger formation despite normal ligand binding affinities. Dominant inheritance of the disorder suggests that the mutation produces a dominant-negative effect by an unknown mechanism. Two isoforms of the human TXA2 receptor with different C-terminals have been cloned, TXR-a and TXR-b, both expressed in human platelets^120,121 . The 2 isoforms show similar ligand-binding characteristics and phospholipase C activation but regulate adenylyl cyclase activity in opposite directions: TXR-a activates adenylyl cyclase, while TXR-b inhibits it. The R60L mutation of TXR-a impairs phospholipase C and adenylyl cyclase stimulation, whereas TXR-b with the same mutation retained its ability to inhibit adenylyl cyclase ( Table 1 ; select the OMIM link). Hence, the interaction between splice variants and polymorphisms determines the biological activity of the receptor.

P2Y ₁₂ ADP receptor

Another example of a rare bleeding disorder involving ADP receptors led to the cloning of the elusive Gi-linked P2Y12 receptor and the discovery of a 2-nucleotide deletion in a region mapping to the end of TMD6, associated with the disorder in an affected family¹²² . This ADP receptor subtype was then shown to be the target for antithrombotic drugs such as ticlopidine and clopidogrel. In this fashion, the cloning of a gene causing an inherited disorder can serve in the discovery of new therapeutic agents targeted toward this receptor.

Chemokine receptors

Of considerable current interest are sequence variants of chemokine receptors¹⁸⁵ . At least 2 of these (LESTR/fusin and CKR5) have been identified as coreceptors for cellular entry of HIV^186,187 . Similarly, certain chemokines were found to block HIV entry into cells^188,189 , presumably by competing with the virus for binding to the chemokine receptor. Hence, natural resistance to HIV infection could occur either by high endogenous levels of chemokines or by mutations of the receptors. Indeed, Samson et al¹¹⁰ discovered that a 32 bp deletion in CCR5 with high allele frequency in a Caucasian population (0.092), leading to a frame shift and a nonfunctional protein, appeared to protect homozygous carriers against HIV infection and blocked HIV entry into macrophages lacking functional CCR5. Furthermore, Val64 substitution with Ile was shown to result in heterodimerization of CCR2 with CCR5 or CXCR4, thereby promoting resistance to AIDS^101,104,105 . On the other hand, certain CCR5 and CX3CR1 alleles may be correlated with AIDS progression^109,112 . However, in a subsequent communication extending the studies on CX3CR1 and AIDS progression, a group of investigators failed to confirm an association with receptor polymorphisms and concluded that the results "do not support a clear and consistent role for CX3CR1 in HIV pathogenesis"¹³³ .

Virally induced or encoded receptors

The virus-GPCR nexus turns out to be pervasive. Epstein-Barr virus induces the expression of human GPCRs (EBI 1 and EBI 2) in B-lymphocytes as possible mediators of EBV effects¹¹³ . On the other hand, virally encoded GPCRs-apparently hijacked from mammalian genomes-appear to function as essential promoters of infection. For example, the CMV (cytomegalovirus)-encoded GPCR, US28, is a functional b-chemokine receptor¹¹⁴ and serves also as a coreceptor for HIV-1 entry¹¹⁵ . In yet another twist of the viral plot, Kaposi's sarcoma-associated herpesvirus harbors 4 genes that mimic the cytokine signaling pathway at various junctions, including genes homologous to the chemokines MIP and IL-6¹⁹⁰ . These examples reveal a complex interplay between the genome of the virus and the host. A viral GPCR gene harbored by Kaposi's sarcoma-associated herpesvirus displays spontaneous activity and serves as a viral oncogene and angiogenesis activator¹¹⁶ . This provides yet another example of a GPCR as mediator of viral effects, in this case those of Kaposi's sarcoma. Thus, interindividual variability in receptor activity can result from external factors such as viral infections.

Biogenic amine receptors

Numerous polymorphisms/variants have been described for biogenic amine receptors. The R16G substitution in the b2 adrenoceptor has been associated with nocturnal asthma (Figure 5 ), whereas W64R in the b3 receptor-expressed in adipocytes and involved in energy metabolism-is linked with obesity¹⁶ . Because of the pervasive role of adrenergic receptors, specifically the b2 adrenoceptor, in cardiovascular and pulmonary functions, there has been an intense search for receptor gene variants predisposing to disease, including heart failure, hypertension, and asthma Table 1 . However, these earlier studies have relied on the analysis of single nucleotide polymorphisms, whereas the physiological relevance of chromosomally phased multiple SNPs (haplotypes) remained unknown. The study of Drysdale et al¹⁵ has demonstrated clearly the need to consider haplotypes, because multiple SNPs on the same strand of DNA can result in different effects from what would have been expected from independent contributions of each SNP alone. This is particularly relevant to the b2 adrenoceptor because the SNPs identified within this gene have distinct effects on receptor function. We will discuss the relevance of b2 adrenoceptor haplotype in more detail later in the context of pharmacological implications.

Intensive studies have also focused on dopaminergic and serotonergic receptors because of their presumed relevance to mental disorders ( Table 1 ). However, linking sequence variations of multiple dopamine and serotonin receptors to mental disease has proven difficult at best. Several possible associations between single nucleotide variants and specific disorders are listed in Table 1 . We have already discussed some of the reasons for this lack of clear association linking receptor variants to disease-this topic is not the main focus of the present review. Lack of strong penetrance and multigenic disease origin are the main complicating factors, along with limited ability to classify the phenotype accurately.

Activating mutations

For several receptors, single nucleotide variants have been reported to lead to activation rather than to inactivation. For example, whereas several inactivating point mutations of the calcium sensing receptor cause familial hypercalcemias, different activating mutations result in hypocalcemias^123-128 . Most of these mutations reside in the large N-terminus, the Ca²⁺ -binding module related to periplasmic binding proteins¹⁴⁷ (Figure 3 ). Because this soluble protein module has a well-defined structure and shares homology with numerous other GPCRs fused to it, we might expect similar, yet-to-be-discovered mutations to occur throughout this receptor subgroup.

Activating mutations are likely to be dominant; thus, a single allele expressing a constitutively active receptor is sufficient and can have profound pathophysiological effects^146,163 . Basally active TSH receptor variants cause thyroid adenomas; the D619G and A623I variants are somatic mutations⁸⁷ . Further receptor mutants that signal in the absence of agonists include the parathyroid hormone receptor^77,78 and rhodopsin^129-132 . As pointed out previously, these mutations can occur in various regions of the receptor protein. Of particular interest is the Lys 296 mutation of rhodopsin, which abrogates an ionic bridge to the counterion in TMD3, thereby allowing the receptor to assume an active conformation^131,132 . The result is retinitis and, eventually, blindness. This finding supports the notion that GPCRs are normally constrained in an inactive conformation but can relax into the active conformation after the constraint is released.

Further, spontaneously active GPCRs include a variant of smoothened, which is part of the hedgehog/patched signaling pathway having a key role in basal cell carcinoma. The sonic hedgehog signaling pathway (Shh) proceeds from the soluble Shh to the tumor suppressor patched (PTCH) and the proto-oncogene smoothened (SMO)¹¹⁷ . Whereas SMO is a member of the 7-TMD GPCR class, PTCH is an integral membrane protein with approximately 9 TMDs unrelated in sequence to the GPCRs¹³⁵ . Oncogenic mutations in both PTCH and SMO result in enhanced signaling via the Shh pathway, leading to basal cell carcinoma, medulloblastoma, and other human tumors. Specifically, somatic mutation in SMO has been associated with basal cell carcinoma¹¹⁹ , the most prevalent cancer worldwide, which is caused by ultraviolet irradiation. This led to a search for drugs capable of suppressing the basal activity of SMO, so-called inverse agonists, or antagonists with negative intrinsic activity. Cyclopamine, a plant steroidal alkaloid shown to affect the Shh in embryonic development, suppressed basal and stimulated SMO activity and abnormal cell growth associated with SMO and PTCH oncogenic mutations¹¹⁹ . This provides an intriguing example of a drug discovery taking advantage of activating mutations as a genetic cause of disease. A similar approach may prove valuable for drug discovery targeting activated GPCR variants in general.

The melanocortin receptors MC1-5 have diverse functions throughout the body. With primary location in the skin, the MC1 receptor affects skin pigmentation; receptor variants are associated with skin color⁶⁶ but may also play a role in melanoma⁶⁷ . Multiple variants have also been reported for the MC4 receptor, a recent focus of interest because of its role in appetite suppression, caloric utilization, and body weight⁷⁰ . By integrating signals from melanocortin and Agouti-related protein, an endogenous melanocortin antagonist, MC4 regulates food intake stimuli in the hypothalamus. The presence of endogenous GPCR antagonists is a rare observation; yet, we have found that the opioid peptide dynorphin also serves as an endogenous antagonist, which may regulate melanocortin function under physiological conditions¹⁹¹ . A rare mutation in the MC4 receptor has recently been shown to account for approximately 4% of cases of extreme obesity⁷⁰ . Interestingly, obesity-associated mutations range from inactivating to activating MC4 variants. In contrast, no polymorphisms were associated with morbid obesity in the genes encoding a-MSH or AGRP. Targeting activating variants of MC4 with inverse agonists may lead to therapy of affected individuals.

Spontaneously active wild-type receptors

Spontaneously active variant receptors are to be distinguished from wild-type receptors already endowed with basal signaling activity, including serotonin 5-HT2C, dopamine 1B (D5), B2 bradykinin, MOR, and DOR. The physiological role of basal signaling activity is under debate for these receptor types, but for the histamine H3 receptor, basal signaling contributed to the regulation of histaminergic neurons in vivo⁵⁴ . We will discuss MOR polymorphisms separately as an example of the range of polymorphic effects on receptor function, specifically basal signaling.

In all cases of monogenic Mendelian disorders, sequence variations are rare, and in most cases, treatment options are scarce. Yet, it may be possible to design effective therapies for some of these disorders attributed to variant receptor alleles, particularly by designing inverse agonists (antagonists with intrinsic negative activity) for receptors carrying activating mutations.

Sequence variations of GPCRs and drug effects

Biogenic amine receptors.

Ligand-receptor binding is readily quantified so that a number of variant receptors have been shown to display well-documented altered affinities for their ligands ( Table 1 ). However, a single substitution in the binding pocket may affect only 1 type of ligand and not others. This is indeed the case for the T164I variant of the b2 receptor. Thr164 provides a hydrogen bond to the catechol moiety of adrenaline that is absent in b2 antagonists; hence, this mutation strongly reduces catechol binding without having any effect on antagonist binding^11,13 (Figure 5 ).

Similarly, several single-residue variants of the dopamine D1B receptor selectively affect agonist binding¹⁷ . Variant D2 and D3 receptors may also lead to altered drug response and toxicity-for example, increased tardive dyskinesia caused by antipsychotics. However, none of these variant alleles have been conclusively linked to altered drug response, possibly because the frequency of homozygous carriers is low or because the drug effect is mediated by multiple receptors and penetrance of the variant allele is low.

Recently, a sequence variation (N251K) has been mapped to the i3 loop of the a2A adrenergic receptor¹ . Unlike previously described variants of G protein-coupled receptors, where the minor species causes a loss of function, the phenotype of Lys-251 a2A AR represents a gain of agonist-promoted function. Similarly, a G389R polymorphism in the intracellular cytoplasmic tail near the seventh transmembrane-spanning segment of the human b1 AR leads to a gain of function, enhancing both basal and agonist-stimulated G protein coupling² . Occurring at amino acid position 389, Gly or Arg can be found with allele frequencies of 0.26 and 0.74, respectively; the minor allele was previously considered to be the human wild-type b1 AR.

b2 Adrenoceptor

Altered drug response of variant b2 adrenoceptor ranks among the most cited examples of therapeutic consequences resulting from receptor polymorphisms^3-15 . Several SNPs were shown have profound effects on b2 adrenoceptor function when expressed as single mutations of the wild-type receptor in heterologous cells. A R16G b2 adrenoceptor variant was shown to down-regulate more rapidly upon agonist activation ( Table 1 ). In contrast, a Q27E substitution protects the receptor against down-regulation (Figure 5 )¹¹ . Thus, children with asthma carrying the rather common R16G variant have been suggested to be less responsive clinically to b2 agonists, presumably because the receptor is down-regulated by therapy in vivo^3,4 . However, not all studies have supported this finding, which is based on predictions from in vitro results obtained with b2 adenoceptors containing only a single SNP¹⁵ . Clearly, it is important to consider the haplotype in order to understand the in vivo significance of variant receptor genes. For example, enhanced sensitivity to agonists in individuals with the Q27E b2 receptors (protected from receptor downregulation) may have been expected; however, in the vast majority of cases, R16G and Q27E are located on the same allele-forming a haplotype with 2 sequence variants on the same strand of DNA. It turns out that the R16G substitution overrides the effect of Q27E, causing rapid down-regulation regardless of the presence of the Q27E substitution Figure 5 ) ¹¹ . Recognizing the importance of haplotype, Drysdale et al¹⁵ have determined the b2 adrenoceptor haplotypes of 13 polymorphic sites. Sequence variants included the promoter region of the gene, a T/C allele in the b2 adrenoceptor 5'-leader cistron (b2 adrenoceptor upstream peptide [BUP]), and nonsynonymous SNPs leading to the R16G, Q27D, and T164I substitutions (Figure 5 ). Each of these amino acid substitutions has significant and often opposing effects on receptor function when analyzed in isolation. Of the 8192 possible haplotypes, only 12 were actually found in the study population, and only 4 accounted for the vast majority of all haplotypes ( Table 2 ) ¹⁵ . Comparing homozygous carriers of either 1 of the 2 most common haplotypes (2/2 and 4/4) revealed a significantly increased response (FEV1) to albuterol in patients with asthma having the 2/2 genotype¹⁵ . Allele frequencies differed substantially between various ethnic populations (Table 2 ). These results demonstrate that at least in the case of the b2 adrenoceptor, single allelic sites fail to predict therapeutic outcome-contradicting earlier results-but rather that the combination of SNPs in a haplotype determines the functionality of the receptor¹⁵ . On the basis of these observations, reassessment of the association between variant b2 adrenoceptors and disease outcome, not only in asthma but also in cardiovascular disorders where adrenergic receptors play major roles as well, is needed.

Although b2 adrenoceptor genotyping appears to offer the opportunity of individualized medication, the clinical value remains to be established. Common clinical protocol stipulates that therapy should proceed to alternative drugs such as steroids if b2 agonists are ineffective. Therefore, potential benefits of genotyping remain to be documented for guiding asthma therapy; however, a better understanding of the functional roles of b2 adrenoceptor haplotypes in disease and therapy might prove valuable for determining factors predisposing to disease and optimizing early treatment.

Schizophrenia and clozapine therapy

A number of GPCR variations have been tested for association with schizophrenia, yielding mixed results (33,39,52,53,192). Some association was reported for dopamine D3 and D4 receptor variants, among others, but the penetrance of these variants was marginal. Nevertheless, it is possible that GPCR variants play a significant role in the etiology of schizophrenia. Because most antischizophrenic agents target GPCRs-predominantly dopamine and serotonin receptors^193,194 -it is likely that GPCR variants may also affect the therapeutic response. Altered ligand binding and drug response have been reported for variants of the serotonin 5-HT2A and C receptors (Table 2 )^40-46 . In the case of the 5-HT2A and C receptors, these variants maybe associated with altered response to clozapine in the treatment of schizophrenia. This has been tested in some detail for clozapine by Arranz et al ^41,44 (Figure 6 ). This atypical antipsychotic agent interacts not only with dopamine receptors, the originally intended target, but also with serotonergic, histaminergic, and muscarinic receptors^193-195 , and moreover with ionotropic GABA-A receptors, a unique property of clozapine¹⁹⁶ . To make matters even more complex, clozapine interacts variably with the 5 muscarinic receptor types either as antagonist or partial agonist¹⁹⁵ . Because of this promiscuity, the therapeutically relevant receptor interactions of clozapine remain elusive, although the 5-HT2A and -2C receptors were proposed as a main target⁴¹ . Only 30% to 60% of treated patients respond favorably to clozapine.

To test whether sequence variants can be identified that determine therapeutic outcome with clozapine, Arranz et al⁴⁴ screened a series of polymorphisms in the a2, 5-HT2a, 5-HT2C, and H2 receptors and in the serotonin transporter gene. A combination of 6 polymorphisms resulted in 76% to 77% success in predicting clozapine response⁴⁴ (Figure 6 )-a remarkable result that could presage future clinical applications of pharmacogenetics. These variant alleles involve the genes encoding 5-HT2A and -2C receptors, H2 receptor, and serotonin transporter. However, this analysis not only leaves out several receptors thought to play a role in schizophrenia, it also leaves unclear how the H2 receptor would affect the response and what its role in schizophrenia might be. Therefore, the association of each variant with therapeutic outcome needs to be validated separately before these results can serve in the prediction of therapeutic outcome. Much work remains before genotyping can become useful for optimizing clozapine therapy. Moreover, the results are not readily transferable to other antipsychotic drugs. Antipsychotics and antidepressants are prime candidates for prospective genotyping to select the optimally effective drug because therapeutic response may take weeks to become apparent. Administration of an ineffective drug, therefore, places an undue burden on the patient, both in terms of failure to alleviate symptoms and economics.

Peptide receptors

Protease activated receptor (PAR).

The PAR family includes several receptor subtypes and involves thrombin as 1 of the substrates. The inherent protease activity of the ligand thrombin cleaves the N-terminus, yielding a new N-terminus that serves as a tethered agonist¹⁴⁶ . Recently, a F240S variant of the PAR2 receptor-affecting the second extracellular loop-has been shown to display altered ligand-binding sensitivity⁹⁹ The authors speculate that the F240S allele with a frequency of 0.084 may contribute to, or be predictive of, inflammatory disease.

The µ opioid receptor: multiple sequence variations and multiple effects.

Drug addiction involves a strong genetic component. Whereas addiction generally increases with increasing use, the susceptibility to addiction and its severity appears to be largely determined by genetic factors. MOR is the immediate target and mediator of narcotic addiction; moreover, opioid pathways have been implicated in contributing to drug addiction in general-for example, to alcohol and cocaine-by impinging on dopaminergic pathways to the nucleus accumbens, a central reward locus. This has led to compelling incentives for the study of MOR gene variants as possible contributors to genetic predisposition to addiction. Spread over a fairly large genomic region, the multiexonic MOR harbors numerous SNPs in the coding region as well as in noncoding flanking regions⁹⁶ . None of these variants has been positively linked to narcotic addiction thus far, suggesting that (multiple) other factors play a role in genetic predisposition to drug abuse. However, human MOR variants altering its primary structure (Figure 7 ) have been studied as to their effect on ligand binding. One of the variant MOR receptors, carrying a relatively frequent N40D substitution, displays 2-fold enhanced binding of b-endorphin⁹² . The authors suggest that this change might be relevant to narcotic effects, including addiction; however, it is not clear what, if any, role b-endorphin plays in the process of addiction. An N152D-MOR variant was expressed in reduced quantities upon in vitro transfection, implying some defect in protein folding and trafficking⁹⁵ .

Functional studies on the effect of single SNPs determined in isolation neglects the combined effect of multiple SNPs on the same haplotype, as discussed for the adrenergic receptors. Hoehe et al⁹⁶ analyzed MOR variations in all known functionally relevant regions of the gene, including 6.7 kilobase regulatory, exonic, and partial intronic sequences. They identified 43 sequence variants in 250 cases (individuals with drug dependence) and controls. By applying a statistical approach to deduce the haplotype (ie, the combination of variants on the same strand of DNA), the authors were able to cluster the haplotypes into 2 functionally related categories. One of these was significantly more frequent in substance-dependent individuals of African American descent, but not in other ethnic groups studied. This reveals ethnic admixture as an important factor in such association studies involving complex traits because ethnic populations are likely to carry distinct sets of polymorphisms and haplotypes. As a result, ethnic admixture is a confounding factor in pharmacogenetic studies unless rigorously controlled for. The results also provide another example of how haplotype analysis can serve to identify complex genotype/phenotype relationships. Although potentially of broad significance, these results need to be validated. In a more limited analysis considering the haplotype associated with only 2 SNPs in the coding region of exon I of MOR (leading to A6V and D40N), Gelernter et al¹⁹⁷ were unable to establish either single polymorphisms or the haplotypes as risk factors in alcohol- and drug-dependent subjects. More work is needed to clarify MOR haplotype contributions to drug addiction.

We are investigating functional changes resulting from variations leading to altered primary structure in the i3 loop of MOR (H260R, H265R, S268P), which represent the primary domain for receptor-G protein coupling (Figure 7 ). Hoellt et al⁹⁴ had already demonstrated that the S268P substitution results in a diminution of receptor desensitization, apparently because of the disruption of this CaMK-II phosphorylation consensus site. Whereas this work was performed with a rat-MOR gene, we have obtained a similar result with the human MOR having an identical i3 loop (Wang et al, submitted). On the other hand, Befort et al ⁹⁵ have shown that the S268P variant has a reduced maximal capacity for coupling to G proteins, whereas the H265R variant receptor did not show any obvious effects. We have identified yet another sporadic SNP affecting the i3 loop structure of MOR (D274N), but the functional consequences remain unknown^93a .

We have recently established that the i3 loop of MOR interacts with both Gi/Go coupling proteins and with calmodulin¹⁴³ because of overlapping sequence motifs required for interaction with either of these 2 major second messengers and cellular regulators. Calmodulin appears to interact with MOR at the i3 loop in such a way that it competes with G proteins binding to MOR (Figure 2 ). One important consequence of MOR-calmodulin binding is to reduce basal (spontaneous) signaling of MOR, which we have proposed plays a key role in narcotic addiction^160,161 . Our results clearly demonstrate that calmodulin serves an important function in regulating basal MOR signaling during morphine exposure¹⁴⁴ . Moreover, calmodulin also may serve as a second messenger of MOR; upon receptor stimulation, it is released from the plasma membrane and calmodulin translocates to the nucleus where it regulates CREB (cAMP response element binding protein) phosphorylation¹⁴⁵ -an event thought to contribute to narcotic dependence. Therefore, we were interested in determining the effects of each polymorphic substitution in the i3 loop on both G protein coupling (in particular, basal coupling) and calmodulin binding. The results show that H260R and H265R have low spontaneous basal G protein-coupling activity, whereas H265R- and S268P-MOR are deficient in calmodulin binding (Figure 8 )^93a .

Because these sequence variants are relatively rare (a single allele found in a population sample of 250 individuals), they cannot account for a substantial portion of the genetic predisposition for drug abuse. However, even if rare, these MOR variants might provide new insights into the mechanism underlying narcotic addiction. Conceivably, both spontaneous signaling and calmodulin-MOR interactions might play a significant role in the addictive process. This would permit us to search for candidate genes in diverse signaling pathways or to design novel approaches to therapy of addiction. For example, we have recently identified neutral antagonists that do not suppress the up-regulated basal MOR activity in dependent tissue, observed with naloxone and naltrexone. As predicted from the hypothesis that high basal activity plays a role in narcotic dependence, these neutral antagonists caused significantly reduced withdrawal symptoms in morphine-dependent mice¹⁶⁵ .

Future Directions

Our knowledge about receptor polymorphisms reveals growing insights into the nature and significance of sequence variations in GPCRs. However, because of structural heterogeneity, receptor multiplicity, and redundancy in complex receptor signaling pathways, identifying the relevance of a single receptor variant is difficult. We suspect that receptor signaling may frequently be impaired by variants of downstream signaling molecules, rather than the receptor itself. Moreover, genetic disorders, in particular mental illness and neurodegenerative disorders, are multigenic. We have relied on the study of candidate genes or linkage analysis involving finite chromosomal locations. However, progress has been slow, and a new approach is needed to resolve the main questions-which genes predispose to disease and which are linked to drug response, either desired effect or toxicity. A promising approach to resolving these questions comes from genome-wide linkage studies using SNPs. Clinical drug trials with genome-wide scanning were first started by Genset Co., using DNA-array technology with 60 000 SNPs. The SNP projects of Celera, a consortium of leading drug companies (SNP consortium), and the public genome sequencing effort now have amassed in excess of 3 million SNPs, promising to enhance the power of genome-wide association studies. This approach might eventually enable researchers to pinpoint genes that contribute to complex disease and therapeutic outcomes. This could lead to more efficacious therapy tailored toward small patient populations. Before this scenario can be played out, however, we need to develop novel methodologies for extensive SNP analysis and statistical treatment of the resulting complex data sets. This process could likely take decades before it becomes the mainstream approach of the pharmaceutical industry. However, the beacons guiding these developments have been planted, and a compelling future direction for novel drug therapies is beginning to emerge.

References

1. Small KM, Forbes SL, Brown KM, Liggett SB. An asn to lys polymorphism in the third intracellular loop of the human alpha 2A-adrenergic receptor imparts enhanced agonist-promoted Gi coupling. J Biol Chem. 2000;275:38518-38523. [PUBMED]

2. Mason DA, Moore JD, Green SA, Liggett SB. A gain-of-function polymorphism in a G-protein coupling domain of the human beta1-adrenergic receptor. J Biol Chem. 1999;274:12670-12674. [PUBMED]

3. Turki J, Pak J, Green S, Martin R, Liggett SB. Genetic polymorphisms of the b2-adrenergic receptor in nocturnal and non-nocturnal asthma. Evidence that Gly 16 correlates with the nocturnal phenotype. J Clin Invest. 1995;95:1635-1641. [PUBMED]

4. Martinez FD, Graves PE, Baldini M, Solomon S, Erickson R. Association between genetic polymorphisms of the b2-adrenoceptor and response to albuterol in children with and without a history of wheezing. J. Clin. Invest. 1997: 100:3184-3188. [PUBMED]

5. Reihsaus E, Innis M, MacIntyre N, Liggett SB. Mutations in the gene encoding for the b2 adrenergic receptor in normal and asthmatic subjects. Am J Respir Cell Mol Biol. 1993;8:334-339. [PUBMED]

6. Bray MS, Krushkal J, Li L, et al. Positional genomic analysis identifies the beta (2)-adrenergic receptor gene as a susceptibility locus for human hypertension. Circulation. 2000;101: 2877-2882. [PUBMED]

7. Wagoner LE, Craft LL, Singh B, et al. Polymorphisms of the beta (2)-adrenergic receptor determine exercise capacity in patients with heart failure. Circ Res. 2000;86:834-840. [PUBMED]

8. Summerhill E, Leavitt SA, Gidley H, Parry R, Solway J, Ober C.. Beta-2 adrenergic receptor polymorphism tied to reduced lung function. Am J Resp Crit Care Med. 2000;162:599-602. [PUBMED]

9. Xu BY, Huang D, Pirskanen R, Lefvert A. Beta2-adrenergic receptor gene polymorphisms in myasthenia gravis (MG). Clin Exp Immunol. 2000;119:156-160. [PUBMED]

10. Dewar JC, Wilkinson J, Wheatley A, et al. The glutamine 27 beta2-adrenoceptor polymorphism is associated with elevated IgE levels in asthmatic families. J Allergy Clin Immunol. 1997;100:261-265. [PUBMED]

11. Green SA, Turki J, Bejarano P, Hall IP, Liggett SB. Influence of beta2-adrenergic receptor genotypes on signal transduction in human airway smooth muscle cells. Am J Respir Cell Mol Biol. 1995;13:25-33. [PUBMED]

12. Meirhaeghe A, Helbecque N, Cottel D, Amouyel P. Beta2-adrenoceptor gene polymorphism, body weight, and physical activity. Lancet. 1999;353:896. [PUBMED]

13. Green SA, Cole G, Jacinto M, Innis M, Liggett SB. A polymorphism of the human b2-adrenergic receptor within the fourth transmembrane domain alters ligand binding and functional properties of the receptor. J Biol Chem. 1993;268:23116-23121. [PUBMED]

14. Birnbaumer M. Mutations and diseases of G protein coupled receptors. J Recept Signal Transduct Res. 1995;15:131-160. [PUBMED]

15. Drysdale CM, McGraw DW, Stack CB, et al. Complex promoter and coding region b2-adrenergic receptor haplotypes alter receptor expression and predict in vivo responsiveness. Proc Natl Acad Sci U S A. 2000;97:10483-10488. [PUBMED]

16. Mitchell BD, Blangero J, Comuzzie AG, et al. A paired sibling analysis of the b-3 adrenergic receptor and obesity in Mexican Americans. J Clin Invest. 1998;101:584-587. [PUBMED]

17. Cravchik A, Gejman PV. Functional analysis of the human D5 dopamine receptor missense and nonsense variants: differences in dopamine binding affinities. Pharmacogenetics. 1999;9:199-206. [PUBMED]

18. Blum K, Sheridan PJ, Wood RC, Braverman ER, Chen TJ, Comings DE. Dopamine D2 receptor gene variants: association and linkage studies in impulsive-addictive-compulsive behaviour. Pharmacogenetics. 1995;5:121-141. [PUBMED]

19. Blum K, Braverman ER, Wood RC, et al. Increased prevalence of the Taq I A1 allele of the dopamine receptor gene (DRD2) in obesity with comorbid substance use disorder: a preliminary report. Pharmacogenetics. 1996;6:297-305. [PUBMED]

20. Blum K, Noble EP, Sheridan PJ, et al. Association of the A1 allele of the D2 dopamine receptor gene with severe alcoholism. Alcohol. 1991;8:409-416. [PUBMED]

21. Hietala J, Pohjalainen T, Heikkila-Kallio U, West C, Salaspuro M, Syvalathi E. Allelic association between D2 but not D1 dopamine receptor gene and alcoholism in Finland. Psychiatr Genet. 1997;7:19-25. [PUBMED]

22. Noble EP. The D2 dopamine receptor gene: a review of association studies in alcoholism and phenotypes. Alcohol. 1998;16:33-45. [PUBMED]

23. Thompson J, Thomas N, Singleton A, et al. D2 dopamine receptor gene (DRD2) Taq1 A polymorphism: reduced dopamine D2 receptor binding in the human striatum associated with the A1 allele. Pharmacogenetics. 1997;7:479-84. [PUBMED]

24. Comings DE, Rosenthal RJ, Lesieur HR, et al. A study of the dopamine D2 receptor gene in pathological gambling. Pharmacogenetics. 1996;6:223-232. [PUBMED]

25. Chen CH, Wei FC, Koong F-J, Hsiao K. Association of Taq1 A polymorphism of dopamine D2 receptor gene and tardive dyskinesia in schizophrenia. Biol Psychiatry. 1997;41:827-829. [PUBMED]

26. Cravchik A, Sibley DR, Gejman PV. Analysis of neuroleptic binding affinities and potencies for the human different D2 dopamine receptor missense variants. Pharmacogenetics. 1999;9:17-23. [PUBMED]

27. Klein C, Brin MF, Kramer P, et al. Association of a missense change in the D2 dopamine receptor with myoclonus dystonia. Proc Natl Acad Sci U S A. 1999;96:5173-5176. [PUBMED]

28. Lannfelt L, Sokoloff P, Martres MP, et al. Amino acid substitution in the dopamine D3 receptor as a useful polymorphism for investigating psychiatric disorders. Psychiatr Genet. 1992;2:249-256.

29. Dikeos DG, Papadimitriou GN, Avramopoulos D, et al. Association between the dopamine D3 receptor gene locus (DRD3) and unipolar affective disorder. Psychiatr Genet. 1999;9:189-195. [PUBMED]

30. Hawi Z, McCabe U, Straub RE, et al. Examination of new and reported data of the DRD3/MscI polymorphism: no support for the proposed association with schizophrenia. Mol Psychiatry. 1998;3:150-155. [PUBMED]

31. Steen VM, Loevlie R, MacEwan T, McCreadie RG. Dopamine D3 receptor variant and susceptibility to tardive dyskinesia in schizophrenic patients. Mol Psychiatry. 1997;2:139-145. [PUBMED]

32. Basile VS, Masellis M, Badri F, et al. Association of the MscI polymorphism of the dopamine D3 receptor gene with tardive dyskinesia in schizophrenia. Neuropsychopharmacology. 1999;21:17-27. [PUBMED]

33. Sinagnanasundadaram S, Morris AG, Gaitonde EJ, McKenna PJ, Mollon JD, Hunt DM. A cluster of single nucleotide polymorphisms in the 5'-leader of the human dopamine D3 receptor gene (DRD3) and its relationship to schizophrenia. Neurosci Lett. 2000;279:13-16. [PUBMED]

34. Van Tol HHM, Wu CM, Guan HC, et al. Multiple dopamine D4 receptor variants in the human population. Nature. 1992;358:149-152. [PUBMED]

35. Newman-Tancredi A, Audinot V, Chaput C, Verriele L, Millan MJ. [35S]Guanosine-5'o-(3-thio)triphosphate binding as a measure of efficacy at human recombinant dopamine D4.4 receptors: actions of antiparkinsonian and antipsychotic drugs. J Pharmacol Exp Ther. 1997;282:181-191. [PUBMED]

36. Gilliland SL, Alper RH. Characterization of dopaminergic compounds at hD2short, hD4.2 and hD2.7 receptors in agonist stimulated [35S]-GTPgammaS binding assays. NS Arch Pharmacol. 2000;361:498-504. [PUBMED]

37. Perez de Castro I, Ibanez A, Torres P, Saiz-Ruiz J, Fernandez -Piqueras J. Genetic association study between pathological gambling and a functional DNA polymorphism at the D4 receptor gene. Pharmacogenetics. 1997;7:345-348. [PUBMED]

38. Liu IS, Seeman P, Sanyal S, et al. Dopamine D4 receptor variant in Africans, D4 valine194glycine, is insensitive to dopamine and clozapine: report of a homozygous individual. Am J Med Genet. 1996;61:277-282. [PUBMED]

39. Okuyama Y, Ishiguro H, Toru M, Arinami T. A genetic polymorphism in the promoter region of DRD4 associated with expression and schizophrenia. Biochem Biophys Res Comm. 1999;258:292-295. [PUBMED]

40. Holmes C, Arranz MJ, Powell JF, Collier DA, Lovestone S. 5-HT2A and 5-HT2C receptor polymorphisms and psychopathology in late onset Alzheimer's disease. Hum Mol Genet. 1998;7: 1506-1509. [PUBMED]

41. Arranz M, Collier D, Sodhi M, et al. Association between clozapine response and allelic variation in 5-HT2A receptor gene. Lancet. 1995;346:281-282. [PUBMED]

42. Joober R, Benkelfat C, Brisebois K, et al. T102C polymorphism in the 5-HT2A gene and schizophrenia: relation to phenotype drug response variability. J Psychiatry Neurosci. 1999;24:141-146. [PUBMED]

43. Murray MJ, Munro J, Sham P, et al. Metaanalysis of studies on genetic variation in 5HT2A receptors and clozapine response. Schiz Res. 1998;32:93-99. [PUBMED]

44. Arranz MJ, Munro J, Bolonna A, et al. Pharmacogenetic prediction of clozapine response. Lancet. 2000;355:1615-1616. [PUBMED]

45. Ozaki N, Lubierman V, Lu SJ, Lappalainen J, Rosenthal NE, Goldman D. A naturally occurring amino acid substitution of the human serotonin 5HT2A receptor influences amplitude and timing of intracellular calcium mobilization. J Neurochem. 1997;68:2186-2193. [PUBMED]

46. Nacmias B, Ricca V, Tedde A, Mezzani B, Rotella CM, Sorbi S. 5-HT2A receptor gene polymorphisms in anorexia nervosa and bulimia nervosa. Neurosci Lett. 1999;277:134-136. [PUBMED]

47. Sodhi MS, Arranz MJ, Curtis D, et al. Association between clozapine response and allelic variation in the 5-HT2C receptor gene. Neuroreport. 1995;7:169-172. [PUBMED]

48. Yuan X, Ishiyama-Shigemoto S, Koyama W, Nonaka K. Identification of polymorphic loci in the promoter region of the serotonin 5HT2C receptor and their association with obesity and type II diabetes. Diabetologica. 2000;43:373-376. [PUBMED]

49. Bruss M, Bonisch H, Buhlen M, Nothen MM, Propping P, Gothert M. Modified ligand binding to the naturally occurring Cys-124 variant of the human serotonin 5-HT1B receptor. Pharmacogen. 1999;9:1:95-102. [PUBMED]

50. Tsai SJ, Liu HC, Liu TY, Wang YC, Hong CJ. Association analysis of the 5-HT6 receptor polymorphism C267T in Alzheimer's disease. Neurosci Lett. 1999;276:138-139. [PUBMED]

51. Sasaki Y, Ihara K, Ahmed S, et al. Lack of association between atopic asthma and polymorphisms of the histamine H1 receptor, histamine H2 receptor, and N-methyltransferase genes. Immunogenetics. 2000;51:238-240. [PUBMED]

52. Orange PR, Heath PR, Wright SR, Ramchand CM, Kolkeivicz L, Pearson RC. Individuals with schizophrenia have an increased incidence of the H2R649G allele for the histamine H2 receptor. Mol Psychiat. 1996;6:466-469.

53. Ito C, Morriset S, Krebs MO, et al. Histamine H2 gene variants: lack of association with schizophrenia. Mol Psychiat. 2000;5:159-164.

54. Morisset S, Rouleau A, Ligneau X, et al. High constitutive activity of native H3 receptors regulates histamine neurons in brain. Nature. 2000;408:860-864. [PUBMED]

55. Rice GI, Foy CA, Grant PJ. Angiotensin converting enzyme and angiotensin II type 1-receptor gene polymorphisms and risk of ischaemic heart disease. Cardiovasc Res. 1999;41:746-753. [PUBMED]

56. van Geel PP, Pinto YM, Zwinderman AH, et al. Increased risk for ischaemic events is related to combined RAS polymorphism. Heart. 2001;85:458-462. [PUBMED]

57. Benetos A, Cambien F, Gautier S, Ricard S, et al. Influence of the angiotensin II type 1 receptor gene polymorphism on the effects of perindopril and nitrendipine arterial stiffness in hypertensive individuals. Hypertension. 1996;28:1081-1084. [PUBMED]

58. van Geel PP, Pinto YM, Buikema H, van Gilst WH. Is the A1166C polymorphism of the angiotensin II type 1 receptor involved in cardiovascular disease? Eur Heart J. 1998;19:G13-G17. [PUBMED]

59. Henrion D, Amant C, Benessiano J, et al.. Angiotensin II type 1 receptor gene polymorphism is associated with an increased vascular reactivity in the human mammary artery in vitro. J Vasc Res. 1998;35:356-362. [PUBMED]

60. Nicaud V, Poirier O, Behague I, et al. Polymorphisms of the endothelin-A and -B receptor genes in relation to blood pressure and myocardial infarction: the Etude Cas-Temoins sur l'Infarctus du Myocarde (ECTIM) Study. Am J Hypertens. 1999;12:304-310. [PUBMED]

61. Puffenberger EG, Hosoda K, Washington SS, et al. A missense mutation of the endothelin-B receptor gene in multigenic Hirschsprung's disease. Cell. 1994;79:1257-1266. [PUBMED]

62. Osuga Y, Hayashi M, Kudo M, Conti M, Kobilka B, Hsueh AJ. Co-expression of defective luteinizing hormone receptor fragments partially reconstitutes ligand-induced signal generation. J Biol Chem. 1997;272:25006-25012. [PUBMED]

63. Shenker A, Laue L, Kosugi S, Merendino JJ, Minegishi T, Cutler GB. A constitutively activating mutation of the luteinizing hormone receptor in familial male precocious puberty. Nature. 1993;365:652-354. [PUBMED]

64. Evans BA, Bowen DJ, Smith PJ, Clayton PE, Gregory JW. A new point mutation in the luteinising hormone receptor gene in familial and sporadic male limited precocious puberty: genotype does not always correlate with phenotype. J Med Genet. 1996;33:143-147. [PUBMED]

65. Gromoll J, Simoni M, Norhoff V, Behre HM, De Geyter C, Nieschlag E. Functional and clinical consequences of mutations in the FSH receptor. Mol Cell Endocrin. 1996;125:177-182. [PUBMED]

66. Valverde P, Healy E, Jackson I, Rees JL, Thody AJ. Variants of the melanocyte-stimulating hormone receptor gene are associated with red hair and fair skin in humans. Nature Gen. 1995;11:328-330. [PUBMED]

67. Valverde P, Healy E, Sikkink S, et al. The Asp84Glu variant of the melanocortin 1 receptor (MC1R) is associated with melanoma. Hum Mol Gen. 1996;5:1663-1666. [PUBMED]

68. Xu X, Thornwall M, Lundin LG, Chhajlani V. Val92Met variant of the melanocyte stimulating hormone receptor gene. Nat Genet. 1996;14:384. [PUBMED]

69. Koppula SV, Robbins LS, Lu D, et al. Identification of a common polymorphism in the coding sequence of the human MSH receptor (MC1R) with possible biological effects. Hum Mutat. 1997;9:30-36. [PUBMED]

70. Vaisse C, Clement K, Durant E, Hercberg S, Guy-Grand B, Froguel P. Melanocortin-4 mutations are frequent and heterogeneous cause of morbid obesity. J Clin Invest. 2000;106:253-262. [PUBMED]

71. Hinney A, Schmidt A, Nottebom K, et al. Several mutations in the melanocortin-4 receptor gene including a nonsense and a frameshift mutation associated with dominantly associated obesity in humans. J Clin Endocrinol Metab. 1999;84:1483-1486. [PUBMED]

72. Tsigos C, Arai K, Hung W, Chrousos GP. Hereditary isolated glucocorticoid deficiency is associated with abnormalities of the adrenocorticotropin receptor gene. J Clin Invest. 1993;92:2458-2461. [PUBMED]

73. Clark AJ, McLoughlin L, Grossman A. Familial glucocorticoid deficiency associated with point mutation in the adrenocorticoid receptor. Lancet. 1993;341:461-462. [PUBMED]

74. Weber A, Kapas S, Hinson J, Grant DB, Grossman A, Clark AJ. Functional characterization of the cloned human ACTH receptor: impaired responsiveness of a mutant receptor in familial glucocorticoid deficiency. Biochem Biophys Res Comm. 1993;197:172-178. [PUBMED]

75. Tsigos C, Arai K, Latronico AC, DiGeorge AM, Rapaport R, Chrousos GP. A novel mutation of the adrenocorticotropin receptor (ACTH-R) gene in a family with the syndrome of isolated glucocorticoid deficiency, but no ACTH-R abnormalities in two families with the triple A syndrome. J Clin Endocrinol Metab. 1995;80:2186-2189. [PUBMED]

76. Reincke M, Mora P, Beuschlein F, Arlt W, Chrousos GP, Allolio B. Deletion of the adrenocorticotropin receptor gene in human adrenocortical tumors: implications for tumorigenesis. J Clin Endocrinol Metab. 1997;82:3054-3058. [PUBMED]

77. Schipani E, Kruse K, Juppner H. A constitutively active mutant PTH-PTHrP receptor in Jansen-type metaphyseal chondrodysplasia. Science. 1995;268:98-100. [PUBMED]

78. Schipani E, Langman CB, Parfitt AM, et al. Constitutively activated receptors for parathyroid hormone and parathyroid hormone-related peptide in Jansen's metaphysical chondrodysplasia. N Engl J Med. 1996;335:708-714. [PUBMED]

79. Schipani E, Langman C, Hunzelman J, et al. A novel parathyroid hormone (PTH)/ PTH-related peptide receptor mutation in Jansen's metaphyseal chondrodysplasia. J Clin Endocrinol Metab. 1999;84:3052-3057. [PUBMED]

80. Karaplis AC, He B, Nguyen MT, et al. Inactivating mutation in the human parathyroid hormone receptor 1 gene in Blomstrand chondrodysplasia. Endocrinology. 1998;139:5255-5258. [PUBMED]

81. Karperien M, van der Harten HJ, van Schooten R, et al. A frame-shift mutation in the type I parathyroid hormone (PTH)/PTH-related peptide receptor causing Blomstrand lethal osteochondrodysplasia. J Clin Endocrinol Metab. 1999;84 3713-3720. [PUBMED]

82. Jobert AS, Zhang P, Couvineau A, et al. Absence of functional receptors for parathyroid hormone and parathyroid hormone-related peptide in Blomstrand Chondrodysplasia. J Clin Invest. 1998;102:34-40. [PUBMED]

83. Cuddihy RM, Dutton CM, Bahn RS. A polymorphism in the extracellular domain of the thyrotropin receptor is highly associated with autoimmune thyroid disease in females. Thyroid. 1995;5:89-95. [PUBMED]

84. Cuddihy RM, Schaid DS, Bahn RS. Multivariate analysis of HLA loci in conjunction with a thyrotropin receptor codon 52 polymorphism in conferring risk of Graves' disease. Thyroid. 1996;6:261-265. [PUBMED]

85. Kaczur V, Szalai C, Falus A, Nagy Z, Krajczar G, Balazs C. Polymorphism of the 52 triplet gene (nucleotide 253) of the TSH receptor in Basedow-Graves patients and in healthy controls. Orv Hetil. 1997;138:1625-1628. [PUBMED]

86. Gabriel EM, Bergert ER, Grant CS, van Heerden JA, Thompson GB, Morris JC. Germline polymorphism of codon 727 of human thyroid-stimulating hormone receptor is associated with toxic multinodular goiter. J Clin Endocrinol Metab. 1999;84:3328-3335. [PUBMED]

87. Parma J, Duprez L, Van Sande J, Cochaux P, Gervy C, Mockel J, Dumont J, Vassart G. Somatic mutations in the thyrotropin receptor gene cause hyperfunctioning thyroid adenomas. Nature. 1993;365:649-651. [PUBMED]

88. Rosenthal W, Seibold A, Antaramian A, et al. Molecular identification of the gene responsible for congenital nephrogenic diabetes insipidus. Nature. 1992;359:233-235. [PUBMED]

89. Schoneberg T, Yun J, Wenkert D, Wess J. Functional rescue of mutant V2 vasopressin receptors causing nephrogenic diabetes insipidus by a co-expressed receptor polypeptide. EMBO J. 1996;15:1283-1291. [PUBMED]

90. Birnbaumer M, Gilbert S, Rosenthal W. An extracellular congenital nephrogenic diabetes insipidus mutation of the vasopressin receptor reduces cell surface expression, affinity for ligand, and coupling to the Gs/adenylyl cyclase system. Mol Endocrin. 1994;8:886-894. [PUBMED]

91. Birnbaumer M, Gilbert S, Rosenthal W. Nephrogenic diabetes insipidus. A V2 vasopressin receptor unable to stimulate adenylyl cyclase. J Biol Chem. 1993;268:13030-13033. [PUBMED]

92. Bond C, LaForge KS, Tian M, Melia D, Zhang S, et al. Single-nucleotide polymorphism in the human µ opioid receptor gene alters beta-endorphin binding and activity: possible implications for opiate addiction. Proc Nat Acad Sci U S A. 1998;95:9608-9613. [PUBMED]

93. Sander T, Berlin W, Gscheidel N, Wendel B, Janz D, Hoehe MR. Genetic variation of the human µ-opioid receptor and susceptibility to idiopathic absence epilepsy. Epilepsy Res. 2000;39:57-61. [PUBMED]

93a. Single nucleotide polymorphisms in the human µ opioid receptor gene alter G proten coupling and calmodulin binding. Wang D, Quillan JM, Winans K, Lucas JL, Sadee W. J.Biol.Chem., 2001; in press.

94. Koch T, Kroslak T, Mayer P, Raulf E, Hoellt V. Site mutation in the rat µ-opioid receptor demonstrates the involvement of calcium/calmodulin-dependent protein kinase II in agonist-mediated desensitization. J Neurochem. 1997;69:1767-1770. [PUBMED]

95. Befort K, Filliol D, Decaillot FM, Gaveriaux-Ruff C, Hoehe MR, Kieffer BL. A single nucleotide polymorphic mutation in the human µ-opioid receptor severely impairs receptor signaling. J Biol Chem. 2001;276:3130-3137. [PUBMED]

96. Hoehe J, Koepke K, Wendel B, et al. Sequence variability and candidate gene analysis in complex disease: association of µ opioid receptor gene variation with substance dependence. Human Mol Gen. 2000;9:2895-2908.

97. Mayer P, Rochlitz H, Rauch E, et al. Association between d-opioidreceptor gene polymorphism and heroin dependence in man. Neuroreport. 1997;8:2547-2550. [PUBMED]

98. Franke P, Nothen M, Wang T, et al. Human d-opioidreceptor gene and susceptibility to heroin and alcohol dependence. Am J Med Genet. 1999;88:462-464. [PUBMED]

99. Compton SJ, Cairns JA, Palmer KJ, Al-Ani B, Hollenberg MD, Walls AF. A polymorphic protease-activated receptor 2 (PAR2) displaying reduced sensitivity to trypsin and differential responses to PAR agonists. J Biol Chem. 2000;275:39207-39212. [PUBMED]

100. Gwinn MR, Sharma A, De Nardin E. Single nucleotide polymorphisms of the N-formyl peptide receptor in localized juvenile periodontitis. J Periodontol. 1999;70:1194-1201. [PUBMED]

101. Smith MV, Dean M, Carrington M, et al. Contrasting genetic influence of CCR2 and CCR5 variants on HIV-1 infection and disease progression. Science. 1997;277:959-965. [PUBMED]

102. O'Brien TR, McDermott DH, Ioannidis JP, et al. Effect of chemokine receptor gene polymorphisms on the response to potent antiretroviral therapy. AIDS. 2000;14:821-826. [PUBMED]

103. Hizawa N, Yamaguchi E, Furuya K, Jinushi E, Ito A, Kawakami Y. The role of the C-C chemokine receptor 2 gene polymorphism V64I (CCR2-64I) in sarcoidosis in a Japanese population. Am J Respir Crit Care Med. 1999;159:2021-2023. [PUBMED]

104. Mellado M, Rodriguez-Frade JM, Vila-Coro AJ, de Ana AM, Martinez-A C. Chemokine control of HIV-1 infection. Nature. 1999;400:723-724. [PUBMED]

105. Szalai C, Csaszar A, Czinner A, Szabo T, Panczel P, Madacsy L, Falus A. Chemokine receptor CCR2 and CCR5 polymorphisms in children with insulin-dependent diabetes mellitus. Pediatr Res. 1999;46:82-84. [PUBMED]

106. Zimmermann N, Bernstein JA, Rothenberg ME. Polymorphisms in the human CC chemokine receptor-3 gene. Biochim Biophys Acta. 1998;1442:170-6107. [PUBMED]

107. Bream JH, Young HA, Rice N, et al. CCR5 promoter alleles and specific DNA binding factors. Science. 1999;284:223a. [PUBMED]

108. Martin MP, Dean M, Smith MW, et al. Genetic acceleration of AIDS progression by a promoter variant of CCR5. Science. 1998;282:1907-1910. [PUBMED]

109. McDermott DH, Zimmermann PA, Guignard F, Kleeberger CA, Leitman SF, the Multicenter AIDS Cohort Study (MACS), Murphy PM. CCR5 promoter polymorphism and HIV-1 disease progression. Lancet. 1998;352:866-870. [PUBMED]

110. Samson M, Libert F, Doranz BJ,, et al. Resistance to HIV infection in Caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene. Nature. 1996; 382:722-725. [PUBMED]

111. Rabkin CS, Yang Q, Goedert JJ, Nguyen G, Mitsuya H, Sei S. Chemokine and chemokine receptor gene variants and risk of non-Hodgkin's lymphoma in human immunodeficiency virus-1-infected individuals. Blood. 1999;93:1838-1842. [PUBMED]

112. Faure S, Meyer L, Costagliola D, et al. Rapid progression to AIDS in HIV+ individuals with a structural variant of the chemokine receptor CX3CR1. Science. 2000;287:2274-2277. [PUBMED]

113. Birkenbach M, Josefsen Kyalamanchili R, Lenoir G, Kieff E. Epstein-Barr virus induced genes: first lymphocyte-specific G protein-coupled peptide receptors. J Virol. 1993;67:2209-2220. [PUBMED]

114. Gao JL, Murphy PM. Human cytomegalovirus open reading frame US28 encodes a functional b-chemokine receptor. J Biol Chem. 1994;269:28539-28542. [PUBMED]

115. Pleskoff O, Treboute C, Brelot A, Heveker N, Seman M, Alozon M. Identification of a chemokine receptor encode by human cytomegalovirus as a cofactor for HIV-1 entry. Science. 1997;276:1874-1878. [PUBMED]

116. Bais C, Santomasso B, Coso O, et al. G protein-coupled receptor of Kaposi's sarcoma-associated herpesvirus is a viral oncogene and angiogenesis activator. Nature. 1998;391:86-89. [PUBMED]

117. Lam CW, Xie J, To KF, et al. A frequent activated smoothened mutation in sporadic basal cell carcinomas. Oncogene. 1999;18:833-836. [PUBMED]

118. Xie J, Murone M, Luoh SM, et al. Activating smoothened mutations in sporadic basal cell carcinoma. Nature. 1998;391:90-92. [PUBMED]

119. Talpale J, Chen JK, Cooper MK, et al. Effects of oncogenic mutations in smoothened and patched can be reversed by cyclopamine. Nature. 2000;406:1005-1009. [PUBMED]

120. Hirata T, Kakizuka A, Ushikubi F, Fuse I, Okuma M, Narumiya S. Arg60-to-leu mutation of the human thromboxane A2 receptor in a dominantly inherited bleeding disorder. J Clin Invest. 1994;94:1662-1667. [PUBMED]

121. Hirata T, Ushikubi F, Kakizuka A, Okuma M, Narumiya S. Two thromboxane A(2) receptor isoforms in human platelets: opposite coupling to adenylyl cyclase with different sensitivity to arg60-to-leu mutation. J Clin Invest. 1996;97:949-956. [PUBMED]

122. Hollopeter G, Jantzen H-M, Vincent D, et al. Identification of the platelet ADP receptor targeted by antithrombotic drugs. Nature. 2001;409:202-207. [PUBMED]

123. Ward BK, Stuckey BG, Gutteridge DH, Laing NG, Pullan PT, Ratajczak T. A novel mutation (L174R) in the Ca2+-sensing receptor gene associated with familial hypocalciuric hypercalcemia. Hum Mutat. 1997;10:233-235.

124. Aida K, Koishi S, Inoue M, Nakazato M, Tawata M, Onaya T. Familial hypocalciuric hypercalcemia associated with mutation in the human Ca (2+)-sensing receptor gene. J Clin Endocrinol Metab. 1995;80:2594-2598. [PUBMED]

125. Chou Yh, Pollak MR, Brandi ML, et al. Mutations in the human Ca (2+)-sensing receptor gene that cause familial hypocalciuric hypercalcemia. Am J Hum Genet. 1995;56:1075-1079. [PUBMED]

126. Pollak MR, Brown EM, Chou YH, et al. Mutations in the human Ca(2+)-sensing receptor gene cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Cell. 1993;75:1297-1303. [PUBMED]

127. Pollak MR, Brown EM, Estep HL, et al. Autosomal dominant hypocalcemia caused by a Ca (2+)-sensing receptor gene mutation. Nat Genet. 1994;8:303-307. [PUBMED]

128. Pearce SH, Williamson C, Kifor O, et al. A familial syndrome of hypocalcemia with hypocalciuria due to mutations in the calcium-sensing receptor. N Engl J Med. 1996;335:1115-1122. [PUBMED]

129. Rao VR, Cohen GB, Oprian DD. Rhodopsin mutation G90D and a molecular mechanism for congenital night blindness. Nature. 1994;367:639-642. [PUBMED]

130. Dryja TP, Berson EL, Rao VR, Oprian DD. Heterozygous missense mutation in the rhodopsin gene as a cause of congenital stationary night blindness. Nat Genet. 1993;4:280-283. [PUBMED]

131. Robinson PR, Cohen GB, Zhukovsky EA, Oprian DD. Constitutively active mutants of rhodopsin. Neuron. 1992;9:719-725. [PUBMED]

132. Robinson PR, Cohen GB, Zhukovsky EA, Oprian DD. Constitutive activation of opsin: influence of charge at position 134 and size at position 296. Biochemistry. 1993;32:6111-6115. [PUBMED]

133. Venter JC, Adams MD, Myers EW, et al.. The sequence of the human genome. Science. 2001;291:1304-1350. [PUBMED]

134. Lander ES, Linton LM, Birren B et al. International sequencing consortium. Initial sequencing and analysis of the human genome. Nature. 2001;409:860-921. [PUBMED]

135. Graul RC, Sadee W. Evolutionary relationships among G protein-coupled receptors using a clustered database approach. AAPS PharmSci. 2001; 2001; 3 (2) article 12 (https://www.pharmsci.org/scientificjournals/pharmsci/journal/01_12.html).

136. Namba T, Sugimoto Y, Negishi M ,et al. Alternative splicing of C-terminal tail of prostaglandin E receptor subtype EP3 determines G-protein specificity. Nature. 1993;365:166-169. [PUBMED]

137. Gudermann T, Kalkbrenner F, Schultz G. Diversity and selectivity of receptor-G protein interaction. Ann Rev Pharmacol Toxicol. 1996;36:429-459. [PUBMED]

138. Migeon JC, Nathanson NM. Differential regulation of cAMP-mediated gene transcription by m1 and m4 muscarinic acetylcholine receptors. J Biol Chem. 1994;269:9767-9773. [PUBMED]

139. Moro O, Lameh J, Högger P, Sadee W. Hydrophobic amino acid in the i1 loop plays a key role in receptor-G protein coupling. J Biol Chem. 1993;268:22273-22276. [PUBMED]

140. Moro O, Shockley MS, Lameh J, Sadee W. Overlapping multisite domains of the muscarinic cholinergic Hm1 receptor involved in signal transduction and sequestration. J Biol Chem. 1994;269:6651-6655. [PUBMED]

141. Burstein ES, Spalding TA, Brann MR. The second intracellular loop of the m5 muscarinic receptor is the switch which enables G-protein coupling. J Biol Chem. 1998;273:24322-24327. [PUBMED]

142. Heuss C, Gerber U. G-protein-independent signaling by G-protein-coupled receptors. TiNS. 2000;23:469-475. [PUBMED]

143. Wang D, Sadee W, Quillan JM. Calmodulin binding to G protein-coupling domain of opioid receptors. J Biol Chem. 1999;274:22081-22088. [PUBMED]

144. Wang D, Surratt CK, Sadee W. Calmodulin regulation of basal and agonist-stimulated G protein coupling by µ opioid receptors (OP3) in morphine pretreated cells. J Neurochem. 2000;75:763-771. [PUBMED]

145. Wang D, Tolbert LM, Carlson KW, Sadee W. Nuclear Ca2+/calmodulin translocation activated by µ opioid (OP3) receptor. J Neurochem. 2000;74:1418-1425. [PUBMED]

146. Coughlin SR. Expanding horizons for receptors coupled to G proteins: diversity and disease. Curr Op Cell Biol. 1994;6:191-197. [PUBMED]

147. Felder CB, Graul RC, Lee AY, Merkle HP, Sadee W. Venus flytrap of periplasmic binding proteins: an ancient protein module present in multiple drug receptors. AAPS PharmSci. 1999; 1(2): article 2 https://www.pharmsci.org/scientificjournals/pharmsci/journal/venus/index.html.

148. Chen J, Ishii M, Wang L, Ishii K, Coughlin SR. Thrombin receptor activation. Confirmation of the intramolecular tethered liganding hypothesis and discovery of an alternative intermolecular liganding mode. J Biol Chem. 1994;269:16041-16045. [PUBMED]

149. Law PY, Wong YH, Loh HH. Mutational analysis of the structure and function of opioid receptors. Biopolymers. 1999;51:440-455. [PUBMED]

150. Befort K, Tabbara L, Kling D, Maigret B, Kieffer BL. Role of aromatic transmembrane residues of the delta-opioid receptor in ligand recognition. J Biol Chem. 1996;271:10161-10168. [PUBMED]

151. Lefkowitz RJ, Cotecchia S, Samama P, Costa T. Constitutive activity of receptors coupled to guanine nucleotide regulatory proteins. TiPS. 1993;14:303-307. [PUBMED]

152. Leff P. The two-state model of receptor activation. TiPS. 1995;16:89-97. [PUBMED]

153. Milligan G, Bond RA. Inverse agonism and the regulation of receptor number. TiPS. 1997;18:468-474. [PUBMED]

154. Chidiac P, Hebert TE, Valiquette M, Dennis M, Bouvier M. Inverse agonist activity of beta-adrenergic antagonists. Mol Pharmacol. 1994;45:490-499. [PUBMED]

155. Barker EL, Westphal RS, Schmidt D, Sanders-Bush E. Constitutively active 5-hydroxytryptamine2C receptors reveal novel inverse agonist activity of receptor ligands. J Biol Chem. 1994;269:11687-11690. [PUBMED]

156. Brys R, Josson K, Castelli MP, et al. Reconstituting the human 5-HT1D receptor-G protein coupling: evidence for constitutive activity and multiple receptor conformations. Mol Pharmacol. 2000;57:1132-1141. [PUBMED]

157. Leeb-Lundberg LM, Mathis SA, Herzig MC. Antagonists of bradykinin that stabilize a G-protein-uncoupled state of the B2 receptor act as inverse agonists in rat myometrial cells. J Biol Chem. 1994;269:25970-25973. [PUBMED]

158. Costa T, Herz A. Antagonists with negative intrinsic activity at delta opioid receptors coupled to GTP-binding proteins. Proc Natl Acad Sci U S A. 1989;86:7321-7325. [PUBMED]

159. Jakubik J, Bacakova L, el-Fakahany EE, Tucek S. Constitutive activity of the M1-M4 subtypes of muscarinic receptors in transfected CHO cells and of muscarinic receptors in the heart cells revealed by negative antagonists. FEBS Lett. 1995;377:275-279. [PUBMED]

160. Burford NT, Wang D, Sadee W. G protein coupling of µopioid receptors (OP3): elevated basal signaling activity. Biochem J. 2000;348:531-537. [PUBMED]

161. Wang Z, Bilsky EJ, Porreca F, Sadee W. Constitutive µ receptor activation as a regulatory mechanism underlying narcotic tolerance and dependence. Life Sci. 1994;54:PL 339-350. [PUBMED]

162. Högger P, Shockley MS, Lameh J, Sadee W. Activating and inactivating mutations in N- and C-terminal loop junctions of muscarinic acetylcholine Hm1 receptors. J Biol Chem. 1995;270:7405-7410. [PUBMED]

163. Rao VR, Oprian DD. Activating mutations of rhodopsin and other G protein-coupled receptors. Ann Rev Biophys Biomol Struct. 1996;25:287-314. [PUBMED]

164. Claeysen S, Sebben M, Becamel C, et al. Pharmacological properties of 5-hydroxytryptamine(4) receptor antagonists on constitutively active wild-type and mutated receptors. Mol Pharmacol. 2000;58:136-144. [PUBMED]

165. Wang D, Raehal KM, Bilsky EJ, Sadee W. Inverse agonists and neutral antagonists at µ opioid receptor (MOR): possible role of basal receptor signaling in narcotic dependence. J Neurochem. 2001; 77: 1590-1600. [PUBMED]

166. Allen LF, Lefkowitz RJ, Caron MG, Cotecchia S. G-protein-coupled receptor genes as protooncogenes: constitutively activating mutations of the a1B-adrenergic receptor enhances mitogenesis and tumorigenicity. Proc Natl Acad Sci U S A. 1991;88:11354-11358. [PUBMED]

167. Robb S, Cheek TR, Hannan FL, Hall LM, Midgley JM, Evans PD. Agonist-specific coupling of a cloned Drosophila octopamine/tyramine receptor to multiple second messenger systems. EMBO J. 1994;13:1325-1330. [PUBMED]

168. Perez DM, Hwa J, Gaivin R, Mathur M, Brown F, Graham RM. Constitutive activation of a single effector pathway: evidence for multiple activation states of a G protein-coupled receptor. Mol Pharmacol. 1996;49:112-122. [PUBMED]

169. Houston DB, Howlett AC. Differential receptor-G protein coupling evoked by dissimilar cannabinoid receptor agonists. Cell Signal. 1998;10:667-674. [PUBMED]

170. Arden JR, Segredo V, Wang Z, Lameh J, Sadee W. Phosphorylation and agonist specific intracellular trafficking of an epitope-tagged µ opioid receptor expressed in HEK293 cells. J Neurochem. 1995;65:1636-1641. [PUBMED]

171. Keith DE, Murray SR, Zaki PA, et al. Morphine activates opioid receptors without causing their rapid internalization. J Biol Chem. 1996;271:19021-19024. [PUBMED]

172. Thomas WG, Alan H, Chang C-S, Karnik S. Agonist induced phosphorylation of the angiotensin II (AT1A) receptor requires generation of a conformation that is distinct from the inositol phosphate signaling state. J Biol Chem. 2000;275:2893-2900. [PUBMED]

173. Standifer KM, Clark JA, Pasternak GW. Modulation of µ1 opioid binding by magnesium: evidence for multiple receptor conformations. J Pharmacol Exp Ther. 1993;266:106-1 [PUBMED]

174. Wreggett KA, Wells JW. Cooperativity in the binding properties of purified cardiac muscarinic receptors. J Biol Chem. 1995;270:22499-22499. [PUBMED]

175. Ostrom RS, Post SR, Insel PA. Stoichiometry and compartmentation in G protein-coupled receptor signaling: implications for therapeutic interventions involving Gs. J Pharmacol Exp Ther. 2000;294:407-4 [PUBMED]

176. Rodbell M. The role of GTP-binding proteins in signal transduction: from the sublimely simple to the conceptually complex. Curr Top Cell Regul. 1992;32:1-47. [PUBMED]

177. Jordan BA, Devi LA. G-protein-coupled receptor heterodimerization modulates receptor function. Nature. 1999;399:697-700. [PUBMED]

178. George SR, Fan T, Xie Z, et al. Oligomerization of µ and d-opioidreceptors. Generation of novel functional properties. J Biol Chem. 2000;275:26128-26135. [PUBMED]

179. Rocheville M, Lange DC, Kumar U, Patel SC, Patel RC, Pate YC. Receptors for dopamine and somatostatin: formation of hetero-oligomers with enhanced functional activity. Science. 2000;288:154-157. [PUBMED]

180. Jones KA, Borowsky B, Tamm JA, et al. GABAB receptors function as a heteromeric assembly of the subunits GABABR1 and GABABR2. Nature. 1998;396:674-679. [PUBMED]

181. Liu F, Wan Q, Pristupa ZD, Yu XM, Wang YT, Niznik HB. Direct protein-protein coupling enables cross-talk between dopamine D5 and g acid A receptors. Nature. 2000;403:274-280. [PUBMED]

182. Shenker A. G protein-coupled receptor structure and function: the impact of disease-causing mutations. Baillieres Clin Endocrinol Metab. 1995;9:427-451. [PUBMED]

183. Spiegel AM. Defects in G protein-coupled signal transduction in human disease.Annu Rev Physiol. 1996;58:143-170. [PUBMED]

184. Kamsteeg EJ, Deen PM, van Os CH. Defective processing and trafficking of water channels in nephrogenic diabetes insipidus. Exp Nephrol. 2000;8:326-331. [PUBMED]

185. Kopin AS, McBride EW, Schaffer K, Beinborn M. CCK receptor polymorphisms: an illustration of emerging themes in pharmacogenomics. TiPS. 2000;21:346-353. [PUBMED]

186. Feng Y, Broder CC, Kennedy PA, Berger EA. HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science. 1996;272:872-877. [PUBMED]

187. Alkhatib G, Combadiere C, Broder CC, et al. CC CKR5: A RANTES, MIP-1aa, MIP-1b receptor as a fusion cofactor for macrophage-tropic HIV-1. Science. 1996;272:1955-1958. [PUBMED]

188. Bleul CC, Farzan M, Choe H, et al. The lymphocyte chemoattractant SDF-1 is a ligand for LESTR/fusin and blocks HIV entry. Nature. 1996;382:829-833. [PUBMED]

189. Cocchi F, DeVico AL, Garzino-Demo A, Arya SK, Gallo RC, Lusso P. Identification of RANTES, MIP-1aa, and MIP-1b as the major HIV-suppressive factors produced by CD8+ T cells. Science. 1995;270:1811-1815. [PUBMED]

190. Moore PS, Boshoff C, Weiss RA, Chang Y. Molecular mimicry of human cytokine and cytokine response pathway genes by KSWHV. Science. 1996;274:1739-1743. [PUBMED]

191. Quillan JM, Sadee W. Dynorphin peptides: antagonists at melanocortin receptors. Pharm Res. 1997;14:713-719. [PUBMED]

192. Pickar D. Pharmacogenomics of psychiatric disorders. TiPS. 2001;22:75-83. [PUBMED]

193. Lahti RA, Evans DL, Stratman NC, Figur LM. Dopamine D4 versus D2 receptor selectivity of dopamine receptor antagonists: possible therapeutic implications. Eur J Pharmacol. 1993;236:483-486. [PUBMED]

194. Phillips ST, de Paulis T, Baron BM, et al. Binding of 5H-dibenzo[b,e][1.4]diazepine and chiral 5Hdibenzo[a,d]cycloheptene analogues of clozapine to dopamine and serotonin receptors. J Med Chem. 1994;37:2686-2696. [PUBMED]

195. Olianas MC, Maullu C, Onali P. Mixed agonist-antagonist properties of clozapine at different human cloned muscarinic receptor subtypes expressed in Chinese hamster ovary cells. Neuropsychopharmacology. 1999;20:263-270. [PUBMED]

196. Korpi ER, Wong G, Lueddens H. Subtype specificity of gamma-aminobutyric acid type A receptor antagonism by clozapine. NS Arch Pharmacol. 1995;352:365-373. [PUBMED]

197. Gelernter J, Kranzler H, Cubells J. Genetics of two µopioid receptor gene (OPRM1) exon I polymorphisms: population studies and allele frequencies in alcohol- and drug-dependent subjects. Mol Psychiatr. 1999;4:476-483. [PUBMED]