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PubMed1996

G-protein-coupled receptor regulation: role of G-protein-coupled receptor kinases and arrestins.

G-protein-coupled receptors (GPCRs) represent a large family of proteins that transduce extracellular signals to the interior of cells. Signalling through these receptors rapidly desensitized primarily as the consequence of receptor phosphorylation, but receptor sequestration and downregulation can also contribute to this process. Two families of serine/threonine kinases, second messenger dependent protein kinases and receptor-specific G-protein-coupled receptor kinases (GRKs), phosphorylate GPCRs and thereby contribute to receptor desensitization. Receptor-specific phosphorylation of GPCRs promotes the binding of cytosolic proteins referred to as arrestins, which function to further uncouple GPCRs from their heterotrimeric G-proteins. To date, the GRK protein family consists of six members, which can be further classified into subgroups according to sequence homology and functional similarities. The arrestin protein family also comprises six members, which are subgrouped on the basis of sequence homology and tissue distribution. While the molecular mechanisms contributing to GPCR desensitization are fairly well characterized, little is known about the mechanism(s) by which GPCR responsiveness is reestablished, other than that receptor sequestration (internalization) might be involved. The goal of the present review is to overview current understanding of the regulation of GPCR responsiveness. In particular, we will review new evidence suggesting a pleiotropic role for GRKs and arrestins in the regulation of GPCR responsiveness. GRK-mediated phosphorylation and arrestin binding are not only involved in the functional uncoupling of GPCRs but they are also intimately involved in promoting GPCR sequestration and as such likely play an important role in mediating the subsequent resensitization of GPCRs.

Animals↗
PubMed2006

Cross-talk between G protein-coupled receptor and epidermal growth factor receptor signaling pathways contributes to growth and invasion of head and neck squamous cell carcinoma.

G protein-coupled receptors (GPCR) and the epidermal growth factor receptor (EGFR) are often both overexpressed and contribute to the growth of cancers by activating autocrine pathways. GPCR ligands have been reported to trigger EGFR signaling via receptor cross-talk in cancer cells. Here, we show that GPCR ligands prostaglandin E2 (PGE2) and bradykinin (BK) activate EGFR signaling. Inhibition of EGFR using several strategies, including small-molecule inhibitors and an EGFR-specific antibody, resulted in partial attenuation of signaling downstream of EGFR. PGE2 and BK triggered EGFR signaling by increasing selective autocrine release of transforming growth factor-alpha (TGF-alpha). Inhibition of tumor necrosis factor-alpha-converting enzyme abrogated BK- or PGE2-mediated activation of EGFR signaling. Both PGE2 and BK stimulated head and neck squamous cell carcinoma (HNSCC) invasion via EGFR. Treatment of HNSCC cells with the BK antagonist CU201 resulted in growth inhibition. The combination of CU201 with the EGFR small-molecule inhibitor erlotinib resulted in additive inhibitory effects on HNSCC cell growth in vitro. Inhibition of the PGE2 synthesis pathway with sulindac induced HNSCC cytotoxicity at high doses (EC(50), 620 micromol/L). However, combined inhibition of both EGFR with the tyrosine kinase inhibitor erlotinib and GPCR with sulindac at low doses of 6 and 310 micromol/L, respectively, resulted in synergistic killing of HNSCC tumor cells. Combined blockade of both EGFR and GPCRs may be a rational strategy to treat cancers, including HNSCC that shows cross-talk between GPCR and EGFR signaling pathways.

Animals↗
PubMed2004

G protein-coupled receptor oligomerization: implications for G protein activation and cell signaling.

The cardiovascular system is richly endowed with G protein-coupled receptors (GPCRs), members of the largest family of plasma membrane-localized receptors. During the last 10 years, it has become increasingly clear that many, if not all, GPCRs function in oligomeric complexes, as either homo- or hetero-oligomers. This review explores the mechanistic implications of GPCR dimerization and/or oligomerization on receptor activation and interactions with G proteins. The effects of GPCR oligomerization on receptor pharmacology, GPCR-mediated signaling, and potential contributions to GPCR crosstalk will be considered in the context of receptors important in the cardiovascular system. Our evolving understanding of the structural and functional consequences of GPCR oligomerization may provide novel and more selective sites for pharmacological tuning of cardiovascular function.

Animals↗
PubMed2005

Multi-tasking RGS proteins in the heart: the next therapeutic target?

Regulator of G-protein-signaling (RGS) proteins play a key role in the regulation of G-protein-coupled receptor (GPCR) signaling. The characteristic hallmark of RGS proteins is a conserved approximately 120-aa RGS region that confers on these proteins the ability to serve as GTPase-activating proteins (GAPs) for G(alpha) proteins. Most RGS proteins can serve as GAPs for multiple isoforms of G(alpha) and therefore have the potential to influence many cellular signaling pathways. However, RGS proteins can be highly regulated and can demonstrate extreme specificity for a particular signaling pathway. RGS proteins can be regulated by altering their GAP activity or subcellular localization; such regulation is achieved by phosphorylation, palmitoylation, and interaction with protein and lipid-binding partners. Many RGS proteins have GAP-independent functions that influence GPCR and non-GPCR-mediated signaling, such as effector regulation or action as an effector. Hence, RGS proteins should be considered multifunctional signaling regulators. GPCR-mediated signaling is critical for normal function in the cardiovascular system and is currently the primary target for the pharmacological treatment of disease. Alterations in RGS protein levels, in particular RGS2 and RGS4, produce cardiovascular phenotypes. Thus, because of the importance of GPCR-signaling pathways and the profound influence of RGS proteins on these pathways, RGS proteins are regulators of cardiovascular physiology and potentially novel drug targets as well.

14-3-3 Proteins↗
PubMed2005

S-nitrosothiols modulate G protein-coupled receptor signaling in a reversible and highly receptor-specific manner.

BACKGROUND: Recent studies indicate that the G protein-coupled receptor (GPCR) signaling machinery can serve as a direct target of reactive oxygen species, including nitric oxide (NO) and S-nitrosothiols (RSNOs). To gain a broader view into the way that receptor-dependent G protein activation -- an early step in signal transduction -- might be affected by RSNOs, we have studied several receptors coupling to the Gi family of G proteins in their native cellular environment using the powerful functional approach of [35S]GTPgammaS autoradiography with brain cryostat sections in combination with classical G protein activation assays. RESULTS: We demonstrate that RSNOs, like S-nitrosoglutathione (GSNO) and S-nitrosocysteine (CysNO), can modulate GPCR signaling via reversible, thiol-sensitive mechanisms probably involving S-nitrosylation. RSNOs are capable of very targeted regulation, as they potentiate the signaling of some receptors (exemplified by the M2/M4 muscarinic cholinergic receptors), inhibit others (P2Y12 purinergic, LPA1lysophosphatidic acid, and cannabinoid CB1 receptors), but may only marginally affect signaling of others, such as adenosine A1, mu-opioid, and opiate related receptors. Amplification of M2/M4 muscarinic responses is explained by an accelerated rate of guanine nucleotide exchange, as well as an increased number of high-affinity [35S]GTPgammaS binding sites available for the agonist-activated receptor. GSNO amplified human M4 receptor signaling also under heterologous expression in CHO cells, but the effect diminished with increasing constitutive receptor activity. RSNOs markedly inhibited P2Y12 receptor signaling in native tissues (rat brain and human platelets), but failed to affect human P2Y12 receptor signaling under heterologous expression in CHO cells, indicating that the native cellular signaling partners, rather than the P2Y12 receptor protein, act as a molecular target for this action. CONCLUSION: These in vitro studies show for the first time in a broader general context that RSNOs are capable of modulating GPCR signaling in a reversible and highly receptor-specific manner. Given that the enzymatic machinery responsible for endogenous NO production is located in close proximity with the GPCR signaling complex, especially with that for several receptors whose signaling is shown here to be modulated by exogenous RSNOs, our data suggest that GPCR signaling in vivo is likely to be subject to substantial, and highly receptor-specific modulation by NO-derived RSNOs.

Animals↗
PubMed2005

Genome wide survey of G protein-coupled receptors in Tetraodon nigroviridis.

BACKGROUND: The G-protein-coupled receptors (GPCRs) constitute one of the largest and most ancient superfamilies of membrane proteins. They play a central role in physiological processes affecting almost all aspects of the life cycle of an organism. Availability of the complete sets of putative members of a family from diverse species provides the basis for cross genome comparative studies. RESULTS: We have defined the repertoire of GPCR superfamily of Tetraodon complement with the availability of complete sequence of the freshwater puffer fish Tetraodon nigroviridis. Almost all 466 Tetraodon GPCRs (Tnig-GPCRs) identified had a clear human homologue. 189 putative human and Tetraodon GPCR orthologous pairs could be identified. Tetraodon GPCRs are classified into five GRAFS families, by phylogenetic analysis, concurrent with human GPCR classification. CONCLUSION: Direct comparison of GPCRs in Tetraodon and human genomes displays a high level of orthology and supports large-scale gene duplications in Tetraodon. Examples of lineage specific gene expansions were also observed in opsin and odorant receptors. The human and Tetraodon GPCR sequences are analogous in terms of GPCR subfamilies but display disproportionate numbers of receptors at the subfamily level. The teleost genome with its expanded set of GPCRs provides additional and interesting comparators to study both evolution and function of these receptors.

Animals↗
PubMed2002

The role of beta-arrestins in the termination and transduction of G-protein-coupled receptor signals.

beta-Arrestins are versatile adapter proteins that form complexes with most G-protein-coupled receptors (GPCRs) following agonist binding and phosphorylation of receptors by G-protein-coupled receptor kinases (GRKs). They play a central role in the interrelated processes of homologous desensitization and GPCR sequestration, which lead to the termination of G protein activation. beta-arrestin binding to GPCRs both uncouples receptors from heterotrimeric G proteins and targets them to clathrin-coated pits for endocytosis. Recent data suggest that beta-arrestins also function as GPCR signal transducers. They can form complexes with several signaling proteins, including Src family tyrosine kinases and components of the ERK1/2 and JNK3 MAP kinase cascades. By recruiting these kinases to agonist-occupied GPCRs, beta-arrestins confer distinct signaling activities upon the receptor. beta-arrestin-Src complexes have been proposed to modulate GPCR endocytosis, to trigger ERK1/2 activation and to mediate neutrophil degranulation. By acting as scaffolds for the ERK1/2 and JNK3 cascades, beta-arrestins both facilitate GPCR-stimulated MAP kinase activation and target active MAP kinases to specific locations within the cell. Thus, their binding to GPCRs might initiate a second wave of signaling and represent a novel mechanism of GPCR signal transduction.

Animals↗
PubMed2004

Green fluorescent protein-tagged beta-arrestin translocation as a measure of G protein-coupled receptor activation.

The G protein-coupled receptor (GPCR) superfamily is the largest family of integral membrane proteins. GPCRs respond to a wide variety of sensory and chemical stimuli and contribute directly to the regulation of all major organ systems. As such, GPCRs represent primary drug targets for therapeutic intervention. Although GPCRs respond to a diverse range of ligands and signal through multiple heterotrimeric G proteins, the inactivation of GPCR signaling is mediated by a limited set of proteins. In particular, the desensitization of the majority of GPCRs is mediated by the binding of two arrestin isoforms, beta-arrestin1 and beta-arrestin2, that exhibit overlapping substrate specificity. In response to GPCR activation and phosphorylation by GPCR kinases, beta-arrestins redistribute from the cytosol to the plasma membrane to bind GPCRs and subsequently target the receptors for internalization via clathrin-coated vesicles. This property of beta-arrestins has allowed the development of a green fluorescent protein (GFP)-based assay for detecting GPCR activation by confocal microscopy. This beta-arrestin-GFP translocation methodology is described in detail in this chapter.

Arrestins↗
PubMed2004

Identification of G-protein-coupled receptor phosphorylation sites by 2D phosphopeptide mapping.

Reversible phosphorylation is important for G-protein-coupled receptor (GPCR) signaling, desensitization, and endocytosis, yet the precise location and role of in vivo phosphorylation sites is unknown for most receptors. This chapter describes a powerful analytical method for the direct identification of GPCR phosphorylation sites by two-dimensional (2D) phosphopeptide mapping. The GPCR of interest is isolated from 32P-labeled cells by immunoprecipitation and transferred to nitrocellulose membranes. In situ cleavage by trypsin releases phosphopeptides that are separated by a combination of high-voltage electrophoresis and chromatography. Phosphoamino acid analysis and Edman sequencing of isolated phosphopeptides reveals information that can lead to the direct identification of GPCR phosphorylation sites. Furthermore, the 2D phosphopeptide mapping technique allows the analysis of temporal and positional changes in the GPCR phosphorylation pattern under different physiological conditions.

Animals↗
PubMed2005

Genetic variation in G-protein-coupled receptors--consequences for G-protein-coupled receptors as drug targets.

G-protein-coupled receptors (GPCRs), including 'orphan' GPCRs whose natural ligands are unknown, comprise the largest membrane receptor superfamily and are the most commonly used therapeutic targets. GPCR genetic loci harbour numerous variants, such as DNA insertions or deletions and single nucleotide polymorphisms that alter GPCR expression and function, thereby contributing to inter-individual differences in disease susceptibility/progression and drug responses. In this article, the authors review examples of GPCR genetic variants that influence transcription, translation, receptor folding and expression on cell surface (by affecting receptor trafficking, dimerisation, desensitisation/downregulation), or perturb receptor function (by altering ligand binding, G-protein coupling and receptor constitutive activity). In spite of such effects, assessment for genetic variants is not currently applied to the drug development and approval process or in the clinical use of GPCR drugs. Further insights will, the authors believe, alter drug discovery/development, therapeutics and likely provide new GPCR drug targets.

Drug Delivery Systems↗
PubMed2004

Abnormality of G-protein-coupled receptor kinases at prodromal and early stages of Alzheimer's disease: an association with early beta-amyloid accumulation.

Overwhelming evidence indicates that the effects of beta-amyloid (Abeta) are dose dependent both in vitro and in vivo, which implies that Abeta is not directly detrimental to brain cells until it reaches a threshold concentration. In an effort to understand early Alzheimer's disease (AD) pathogenesis, this study focused on the effects of subthreshold soluble Abeta and the underlying molecular mechanisms in murine microglial cells and an AD transgenic mouse model. We found that there were two phases of dose-dependent Abeta effects on microglial cells: at the threshold of 5 microm and above, Abeta directly induced tumor necrosis factor-alpha (TNF-alpha) release, and at subthreshold doses, Abeta indirectly potentiated TNF-alpha release induced by certain G-protein-coupled receptor (GPCR) activators. Mechanistic studies revealed that subthreshold Abeta pretreatment in vitro reduced membrane GPCR kinase-2/5 (GRK2/5), which led to retarded GPCR desensitization, prolonged GPCR signaling, and cellular hyperactivity to GPCR agonists. Temporal analysis in an early-onset AD transgenic model, CRND8 mice, revealed that the membrane (functional) GRK2/5 in brain cortices were significantly reduced. More importantly, such a GRK abnormality took place before cognitive decline and changed in a manner corresponding with the mild to moderate soluble Abeta accumulation in these transgenic mice. Together, this study not only discovered a novel link between subthreshold Abeta and GRK dysfunction, it also demonstrated that the GRK abnormality in vivo occurs at prodromal and early stages of AD.

Alzheimer Disease↗
PubMed1998

Using green fluorescent protein to understand the mechanisms of G-protein-coupled receptor regulation.

G protein-coupled receptor (GPCR) activation is followed rapidly by adaptive changes that serve to diminish the responsiveness of a cell to further stimulation. This process, termed desensitization, is the consequence of receptor phosphorylation, arrestin binding, sequestration and down-regulation. GPCR phosphorylation is initiated within seconds to minutes of receptor activation and is mediated by both second messenger-dependent protein kinases and receptor-specific G protein-coupled receptor kinases (GRKs). Desensitization in response to GRK-mediated phosphorylation involves the binding of arrestin proteins that serve to sterically uncouple the receptor from its G protein. GPCR sequestration, the endocytosis of receptors to endosomes, not only contributes to the temporal desensitization of GPCRs, but plays a critical role in GPCR resensitization. GPCR down-regulation, a loss of the total cellular complement of receptors, is the consequence of both increased lysosomal degradation and decreased mRNA synthesis of GPCRs. While each of these agonist-mediated desensitization processes are initiated within a temporally dissociable time frame, recent data suggest that they are intimately related to one another. The use of green fluorescent protein from the jellyfish Aqueora victoria as an epitope tag with intrinsic fluorescence has facilitated our understanding of the relative relationship between GRK phosphorylation, arrestin binding, receptor sequestration and down-regulation.

Arrestin↗
PubMed2003

Gene and protein domain-specific patterns of genetic variability within the G-protein coupled receptor superfamily.

INTRODUCTION: Guanine nucleotide binding proteins (G-proteins) represent the targets for >50% of all therapeutics. There is substantial interindividual variation in response to agonists and antagonists directed to these receptors, which may, in part, be due to genetic polymorphisms. As a class, the sequence variability of G-protein-coupled receptor (GPCR) genes has not been characterized. STUDY DESIGN: This variability was investigated by sequencing promoter, 5'- and 3'-UTR, coding blocks, and intron-exon boundaries, of 64 GPCR genes in an ethnically diverse group of 82 individuals. RESULTS: Of the 675 single-nucleotide variations found, 61% occurred in > or =1% of the population sample and the nature of these 412 single nucleotide polymorphisms (SNPs) was assessed. 5'-UTR (p = 0.002) and coding (p = 0.006) SNPs were observed more often in GPCR genes, compared with 309 non-GPCR genes similarly interrogated. The prevalence of non-synonymous coding SNPs was unexpectedly high, with 65% of GPCR genes having at least one. Intron-containing genes had half as many non-synonymous coding SNPs compared with intronless genes (p = 0.0009), suggesting that when introns are not available coding regions provide sites for variation. A distinct relationship between the prevalence of non-synonymous SNPs and receptor structural domains was evident (p = 0.0006 by ANOVA), with variability being most prominent in the transmembrane spanning domains (38%) and the intracellular loops (24%). Phosphoregulatory domains, particularly the carboxy terminus, often the site for agonist-promoted phosphorylation by G-protein coupled receptor kinases, were the least polymorphic (8%). CONCLUSIONS: There is substantial genetic variability in potentially pharmacologically relevant coding and noncoding regions of GPCRs. Such variability should be considered in the development of new agents, or optimization of existing agents, targeted to these receptors.

Analysis of Variance↗
PubMed2001

Peptide-binding G protein-coupled receptors: new opportunities for drug design.

Over the last decades distinct members of the G Protein-Coupled Receptor (GPCR) family emerged as prominent drug targets within pharmaceutical research, since approximately 60 % of marketed prescription drugs act by selectively addressing representatives of that class of transmembrane signal transduction systems. It is noteworthy that the majority of GPCR-targeted drugs elicit their biological activity by selective agonism or antagonism of biogenic monoamine receptors, while the development status of peptide-binding GPCR-addressing compounds is still in its infancy. Exemplified on selected medicinal chemistry projects, this review will focus on the opportunities of therapeutic intervention into a broad spectrum of disease processes through agonizing or antagonizing the functions of peptide-binding GPCRs. In this context, a brief overview of GPCR-mediated signal transduction pathways will be given in order to emphasize the biomedical relevance of a controlled modulation of receptor function. Modern trends on lead finding and optimization strategies for peptide-binding GPCR-targeted low-molecular weight compounds will be highlighted on the basis of current research programs conducted in the areas of angiotensin II, endothelin, bradykinin, neurokinin, neuropeptide Y, LHRH, C5a antagonists, and somatostatin agonists, respectively. Special emphasis will be laid on the elaboration and utilization of structural rationales on the potential drug candidates, thus facilitating more detailed insights into the underlying molecular recognition event.

Animals↗
PubMed2006

Homology modeling of G-protein-coupled receptors and implications in drug design.

G-protein-coupled receptors (GPCRs) are considered therapeutically important due to their involvement in a variety of processes governing several cellular functions, and their tractability as drug targets. A large percentage of drugs on the market, and in development stages, target the super family of the GPCRs. The enormous interest in GPCR drug design is, however, limited by the scarcity of structural information. The only GPCR for which a three dimensional (3D) structure is reported is bovine rhodopsin and it belongs to class A of the GPCR family. As a result, there has been considerable interest in alternative techniques, for example, homology modeling of GPCRs, in order to derive useful three dimensional models of other proteins for use in structure-based drug design. However, homology modeling of GPCRs is not straightforward, and encounters several problems, owing to the availability of a single structural template, as well as the low degree of sequence homology between the template and target sequences. There are several key issues which need to be considered during every stage of GPCR homology modeling, in order to derive reasonable 3D models. Homology modeling of GPCRs has been utilized increasingly in the past few years and has been successful, not only in furthering the understanding of ligand-protein interactions, but also in the identification of new and potent ligands. Thus, with the lessons learned from past experiences and new developments, homology modeling in case of GPCRs can be harnessed for developing more reliable three dimensional models. This, in turn, will provide better tools to use in structure-based drug design leading to the identification of novel and potent GPCR ligands for several therapeutic indications.

Amino Acid Sequence↗
PubMed2005

New prospects for drug discovery from structural studies of rhodopsin.

G-protein-coupled receptors (GPCR) are a major class of membrane proteins belonging to a continuously growing superfamily. These receptors play a critical role in signal transduction, and are among the most important pharmacological drug targets. The first structural model for the GPCR superfamily was the bacterial protein bacteriorhodopsin with its characteristic seven transmembrane (TM) helical architecture. The visual photoreceptor rhodopsin is a better model for GPCR, and the recent elucidation of the crystal structure of bovine rhodopsin has renewed the interest in this receptor as a template for molecular modeling of other GPCR, particularly for the implications in ligand design and drug discovery. In this work different specific structural elements of rhodopsin are reviewed and the role of conserved motifs, like those associated with receptor function, is analyzed. The specific characteristics of the membrane-embedded ligand-binding domain are described. Other aspects, like receptor dimerization or the constitutive activity mechanism, are also outlined. The importance of acquiring knowledge of the active conformation of the receptor by means of both modeling and experimental techniques is also highlighted. In this regard, the model of the activated form of rhodopsin is currently under investigation, and it may provide useful information for pharmaceutical design. Rhodopsin will continue to be a widely used model for GPCR but rhodopsin-based approaches have to be complemented by other theoretical and experimental approaches -while waiting for the crystal structure of other members of the superfamily- if these want to be successfully used for drug discovery.

Drug Design↗
PubMed2001

Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor constitutively activates NF-kappa B and induces proinflammatory cytokine and chemokine production via a C-terminal signaling determinant.

Kaposi's sarcoma-associated herpesvirus (KSHV) is believed to be the causative agent of Kaposi's sarcoma (KS), a multicentric growth factor-dependent tumor common in AIDS patients characterized histopathologically by spindle cell proliferation, angiogenesis, and leukocyte infiltration. Recently, open reading frame 74 of KSHV has been implicated as a major viral determinant of KS. Open reading frame 74 encodes KSHV G protein-coupled receptor (GPCR), a constitutively active chemokine receptor that directly transforms NIH 3T3 cells in vitro and induces multifocal KS-like lesions in KSHV-GPCR-transgenic mice. Interestingly, receptor-positive cells are very rare in lesions from these mice, implicating an indirect mechanism of tumorigenesis. In this regard, here we report that expression of KSHV-GPCR in transfected epithelial, monocytic, and T cell lines induced constitutive activation of the immunoregulatory transcription factors AP-1 and NF-kappaB. This was associated with constitutive induction of the proinflammatory NF-kappaB-dependent cytokines IL-1beta, IL-6, and TNF-alpha, and chemokines monocyte chemoattractant protein-1 and IL-8, as well as the AP-1-dependent basic fibroblast growth factor. In addition, IL-2 and IL-4 production was induced in transfected Jurkat T cells. Truncation of the final five amino acids in the cytoplasmic tail of KSHV-GPCR caused complete loss of its transforming and NF-kappaB-inducing activities, without affecting receptor expression or ligand binding. These data suggest that KS results in part from KSHV-GPCR induction of proinflammatory cytokine and growth factor gene expression, mediated by a signaling determinant within the last five amino acids of the C terminus, a domain that is also critical for direct cell transformation.

3T3 Cells↗
PubMed2000

The effect of mutations in the DRY motif on the constitutive activity and structural instability of the histamine H(2) receptor.

In previous studies we showed that the wild-type histamine H(2) receptor stably expressed in Chinese hamster ovary cells is constitutively active. Because constitutive activity of the H(2) receptor is already found at low expression levels (300 fmol/mg protein) this receptor is a relatively unique member of the G-protein-coupled receptor (GPCR) family and a useful tool for studying GPCR activation. In this study the role of the highly conserved DRY motif in activation of the H(2) receptor was investigated. Mutation of the aspartate 115 residue in this motif resulted in H(2) receptors with high constitutive activity, increased agonist affinity, and increased signaling properties. In addition, the mutant receptors were shown to be highly structurally instable. Mutation of the arginine 116 residue in the DRY motif resulted also in a highly structurally instable receptor; expression of the receptor could only be detected after stabilization with either an agonist or inverse agonist. Moreover, the agonist affinity at the Arg-116 mutant receptors was increased, whereas the signal transduction properties of these receptors were decreased. We conclude that the Arg-116 mutant receptors can adopt an active conformation but have a decreased ability to couple to or activate the G(s)-protein. This study examines the pivotal role of the aspartate and arginine residues of the DRY motif in GPCR function. Disruption of receptor stabilizing constraints by mutation in the DRY motif leads to the formation of active GPCR conformations, but concomitantly to GPCR instability.

Amino Acid Motifs↗