Deorphanization of Dresden G protein-coupled receptor for an odorant receptor

Dresden G protein-coupled receptor (D-GPCR) is one of orphan G protein-coupled receptors (GPCR). Here we report the identification of the ligands and the characterization of D-GPCR. We investigated over 5000 compounds to evoke the response mediated by D-GPCR and identified 3-methyl-valeric acid and 4-methyl-valeric acid as agonists using a cAMP assay. It is of interest that they dramatically enhanced the intracellular cAMP accumulation and the CRE-luciferase activity in CHO-K1 cells and HEK293 cells expressing the chimeric protein of D-GPCR with a rhodopsin-tag at its N-terminus. Our results established new characteristics of D-GPCR as an olfactory receptor. First, agonists of D-GPCR belong to odorants. Second, D-GPCR mRNA is expressed in the olfactory bulb. In addition, D-GPCR was reported to have similar sequences and its genome locus nearby other olfactory receptors. These results suggest D-GPCR is an olfactory receptor.     

Synucleins are a novel class of substrates for G protein-coupled receptor kinases

G protein-coupled receptor kinases (GRKs) specifically recognize and phosphorylate the agonist-occupied form of numerous G protein-coupled receptors (GPCRs), ultimately resulting in desensitization of receptor signaling. Until recently, GPCRs were considered to be the only natural substrates for GRKs. However, the recent discovery that GRKs also phosphorylate tubulin raised the possibility that additional GRK substrates exist and that the cellular role of GRKs may be much broader than just GPCR regulation. Here we report that synucleins are a novel class of GRK substrates. Synucleins (alpha, beta, gamma, and synoretin) are 14-kDa proteins that are highly expressed in brain but also found in numerous other tissues. alpha-Synuclein has been linked to the development of Alzheimer's and Parkinson's diseases. We found that all synucleins are GRK substrates, with GRK2 preferentially phosphorylating the alpha and beta isoforms, whereas GRK5 prefers alpha-synuclein as a substrate. GRK-mediated phosphorylation of synuclein is activated by factors that stimulate receptor phosphorylation, such as lipids (all GRKs) and Gbetagamma subunits (GRK2/3), suggesting that GPCR activation may regulate synuclein phosphorylation. GRKs phosphorylate synucleins at a single serine residue within the C-terminal domain. Although the function of synucleins remains largely unknown, recent studies have demonstrated that these proteins can interact with phospholipids and are potent inhibitors of phospholipase D2 (PLD2) in vitro. PLD2 regulates the breakdown of phosphatidylcholine and has been implicated in vesicular trafficking. We found that GRK-mediated phosphorylation inhibits synuclein's interaction with both phospholipids and PLD2. These findings suggest that GPCRs may be able to indirectly stimulate PLD2 activity via their ability to regulate GRK-promoted phosphorylation of synuclein.     

A G protein-coupled receptor for UDP-glucose

Uridine 5'-diphosphoglucose (UDP-glucose) has a well established biochemical role as a glycosyl donor in the enzymatic biosynthesis of carbohydrates. It is less well known that UDP-glucose may possess pharmacological activity, suggesting that a receptor for this molecule may exist. Here, we show that UDP-glucose, and some closely related molecules, potently activate the orphan G protein-coupled receptor KIAA0001 heterologously expressed in yeast or mammalian cells. Nucleotides known to activate P2Y receptors were inactive, indicating the distinctly novel pharmacology of this receptor. The receptor is expressed in a wide variety of human tissues, including many regions of the brain. These data suggest that some sugar-nucleotides may serve important physiological roles as extracellular signaling molecules in addition to their familiar role in intermediary metabolism.     

G protein-coupled receptor 30 is an estrogen receptor in the plasma membrane

Recently, GPR30 was reported to be a novel estrogen receptor; however, its intracellular localization has remained controversial. To investigate the intracellular localization of GPR30 in vivo, we produced four kinds of polyclonal antibodies for distinct epitopes on GPR30. Immunocytochemical observations using anti-GPR30 antibody and anti-FLAG antibody show that FLAG-GPR30 localizes to the plasma membrane 24 h after transfection. Treatment with estrogen (17beta-estradiol or E2) causes an elevation in the intracellular Ca2+ concentration ([Ca2+]i) within 10 s in HeLa cells expressing FLAG-GPR30. In addition, E2 induces the translocation of GPR30 from the plasma membrane to the cytoplasm by 1 h after stimulation. Immunohistochemical analysis shows that GPR30 exists on the cell surface of CA2 pyramidal neuronal cells. The images on transmission electron microscopy show that GPR30 is localized to a particular region associated with the plasma membranes of the pyramidal cells. These data indicate that GPR30, a transmembrane receptor for estrogen, is localized to the plasma membrane of CA2 pyramidal neuronal cells of the hippocampus in rat brain.     

Peptide substrates for G protein-coupled receptor kinase 2

G protein-coupled receptor kinases (GRKs) control the signaling and activation of G protein-coupled receptors through phosphorylation. In this study, consensus substrate motifs for GRK2 were identified from the sequences of GRK2 protein substrates, and 17 candidate peptides were synthesized to identify peptide substrates with high affinity for GRK2. GRK2 appears to require an acidic amino acid at the −2, −3, or −4 positions and its consensus phosphorylation site motifs were identified as (D/E)X_1–3(S/T), (D/E)X_1–3(S/T)(D/E), or (D/E)X_0–2(D/E)(S/T). Among the 17 peptide substrates examined, a 13-amino-acid peptide fragment of β-tubulin (DEMEFTEAESNMN) showed the highest affinity for GRK2 (K_m, 33.9 μM; V_max, 0.35 pmol min⁻¹ mg⁻¹), but very low affinity for GRK5. This peptide may be a useful tool for investigating cellular signaling pathways regulated by GRK2.     

Evidence for a G protein-coupled gamma-hydroxybutyric acid receptor

gamma-Hydroxybutyric acid (GHB) is a naturally occurring metabolite of GABA that has been postulated to exert ubiquitous neuropharmacological effects through GABA(B) receptor (GABA(B)R)-mediated mechanisms. The alternative hypothesis that GHB acts via a GHB-specific, G protein-coupled presynaptic receptor that is different from the GABA(B)R was tested. The effect of GHB on regional and subcellular brain adenylyl cyclase in adult and developing rats was determined and compared with that of the GABA(B)R agonist (-)-baclofen. Also, using guanosine 5'-O:-(3-[(35)S]thiotriphosphate) ([(35)S]GTPgammaS) binding and low-K:(m) GTPase activity as markers the effects of GHB and (-)-baclofen on G protein activity in the brain were determined. Neither GHB nor baclofen had an effect on basal cyclic AMP (cAMP) levels. GHB significantly decreased forskolin-stimulated cAMP levels by 40-50% in cortex and hippocampus but not thalamus or cerebellum, whereas (-)-baclofen had an effect throughout the brain. The effect of GHB on adenylyl cyclase was observed in presynaptic and not postsynaptic subcellular tissue preparations, but the effect of baclofen was observed in both subcellular preparations. The GHB-induced alteration in forskolin-induced cAMP formation was blocked by a specific GHB antagonist but not a specific GABA(B)R antagonist. The (-)-baclofen-induced alteration in forskolin-induced cAMP formation was blocked by a specific GABA(B)R antagonist but not a specific GHB antagonist. The negative coupling of GHB to adenylyl cyclase appeared at postnatal day 21, a developmental time point that is concordant with the developmental appearance of [(3)H]GHB binding in cerebral cortex, but the effects of (-)-baclofen were present by postnatal day 14. GHB and baclofen both stimulated [(35)S]GTPgammaS binding and low-K:(m) GTPase activity by 40-50%. The GHB-induced effect was blocked by GHB antagonists but not by GABA(B)R antagonists and was seen only in cortex and hippocampus. The (-)-baclofen-induced effect was blocked by GABA(B)R antagonists but not by GHB antagonists and was observed throughout the brain. These data support the hypothesis that GHB induces a G protein-mediated decrease in adenylyl cyclase via a GHB-specific G protein-coupled presynaptic receptor that is different from the GABA(B)R.     

G protein-coupled receptor for diapause hormone, an inducer of Bombyx embryonic diapause

Bombyx diapause hormone was the first chemical substance identified as a maternal control factor that arrests offspring development. However, the molecular mechanisms by which the hormone transduces the signal to the oocyte that induces embryonic diapause immediately after mesoderm segmentation are not fully understood. Here, we describe a cDNA for a G protein-coupled diapause hormone receptor with seven transmembrane domains. Its amino-acid sequence shows a high level of similarity to the receptors of mammalian neuromedin U and insect regulatory peptide, an FXPRL-amide C-terminus. When expressed in a Xenopus oocyte system, the receptor exhibited the highest affinity (EC(50), approximately 70nM) for diapause hormone, when compared with other Bombyx FXPR/KL-amide peptides. Diapause hormone without amidation at the C-terminus, which never induces embryonic diapause in vivo, had no effect in this heterologous expression system. The mRNA is expressed in the ovaries during Bombyx pupal-adult development. These results strongly indicate that the cDNA encodes the diapause hormone receptor.     

A monoclonal antibody for G protein-coupled receptor crystallography

G protein-coupled receptors (GPCRs) constitute the largest family of signaling proteins in mammals, mediating responses to hormones, neurotransmitters, and senses of sight, smell and taste. Mechanistic insight into GPCR signal transduction is limited by a paucity of high-resolution structural information. We describe the generation of a monoclonal antibody that recognizes the third intracellular loop (IL3) of the native human beta(2) adrenergic (beta(2)AR) receptor; this antibody was critical for acquiring diffraction-quality crystals.     

Pharmacological characterization of STKR, an insect G protein-coupled receptor for tachykinin-like peptides

STKR is a G protein-coupled receptor that was cloned from the stable fly, Stomoxys calcitrans. Multiple sequence comparisons show that the amino acid sequence of this insect receptor displays several features that are typical for tachykinin (or neurokinin, NK) receptors. Insect tachykinin-related peptides, also referred to as "insectatachykinins," produce dose-dependent calcium responses in Drosophila melanogaster Schneider 2 cells, which are stably transfected with this receptor (S2-STKR). These responses do not depend on the presence of extracellular Ca(2+)-ions. A rapid agonist-induced increase of inositol 1,4,5-trisphosphate (IP(3)) is observed. This indicates that the agonist-induced cytosolic Ca(2+)-rise is caused by a release of Ca(2+) ions from intracellular calcium stores. The pharmacology of STKR is analyzed by studying the effects of the most important antagonists for mammalian NK-receptors on STKR-expressing insect cells. The results show that spantide II, a potent substance P antagonist, is a real antagonist of insectatachykinins on STKR. We have also tested the activity of a variety of natural insectatachykinin analogs by microscopic image analysis of calcium responses in S2-STKR cells. At a concentration of 1 microM, almost all natural analogs produce a significant calcium rise in stable S2-STKR cells. Interestingly, Stc-TK, an insectatachykinin that was recently discovered in the stable fly (S. calcitrans), also proved to be an STKR-agonist. Stc-TK, a potential physiological ligand for STKR, contains an Ala-residue (or A) instead of a highly conserved Gly-residue (or G). Arch.     

Interacting residues in an activated state of a G protein-coupled receptor

Ste2p, the G protein-coupled receptor (GPCR) for the tridecapeptide pheromone alpha-factor of Saccharomyces cerevisiae, was used as a model GPCR to investigate the role of specific residues in the resting and activated states of the receptor. Using a series of biological and biochemical analyses of wild-type and site-directed mutant receptors, we identified Asn(205) as a potential interacting partner with the Tyr(266) residue. An N205H/Y266H double mutant showed pH-dependent functional activity, whereas the N205H receptor was non-functional and the Y266H receptor was partially active indicating that the histidine 205 and 266 residues interact in an activated state of the receptor. The introduction of N205K or Y266D mutations into the P258L/S259L constitutively active receptor suppressed the constitutive activity; in contrast, the N205K/Y266D/P258L/S259L quadruple mutant was fully constitutively active, again indicating an interaction between residues at the 205 and 206 positions in the receptor-active state. To further test this interaction, we introduced the N205C/Y266C, F204C/Y266C, and N205C/A265C double mutations into wild-type and P258L/S259L constitutively active receptors. After trypsin digestion, we found that a disulfide-cross-linked product, with the molecular weight expected for a receptor fragment with a cross-link between N205C and Y266C, formed only in the N205C/Y266C constitutively activated receptor. This study represents the first experimental demonstration of an interaction between specific residues in an active state, but not the resting state, of Ste2p. The information gained from this study should contribute to an understanding of the conformational differences between resting and active states in GPCRs.     

Endosomal trafficking of the G protein-coupled receptor somatostatin receptor 3

Intracellular trafficking of G protein-coupled receptors (GPCRs) regulates their surface availability and determines cellular response to agonists. Rab GTPases regulate membrane trafficking and identifying Rab networks controlling GPCR trafficking is essential for understanding GPCR signaling. We used real time imaging to show that somatostatin receptor 3 (SSTR3) traffics through Rab4-, Rab21-, and Rab11-containing endosomes, but largely bypasses Rab5 and Rab7 endosomes. We show that SSTR3 rapidly traffics through Rab4 endosomes but moves slower through Rab21 and Rab11 endosomes. SSTR3 passage through each endosomal compartment is regulated by the cognate Rab since expression of the inactive Rab4/S22N, Rab21/T33N, and Rab11/S25N inhibits SSTR3 trafficking. Thus, Rab4, Rab21, and Rab11 may represent therapeutic targets to modulate surface availability of SSTR3 for agonist binding. Our novel finding that Rab21 regulates SSTR3 trafficking suggests that Rab21 may play a role in trafficking of other GPCRs.     

Molecular basis for activation of G protein-coupled receptor kinases

G protein‐coupled receptor (GPCR) kinases (GRKs) selectively recognize and are allosterically regulated by activated GPCRs, but the molecular basis for this interaction is not understood. Herein, we report crystal structures of GRK6 in which regions known to be critical for receptor phosphorylation have coalesced to stabilize the kinase domain in a closed state and to form a likely receptor docking site. The crux of this docking site is an extended N‐terminal helix that bridges the large and small lobes of the kinase domain and lies adjacent to a basic surface of the protein proposed to bind anionic phospholipids. Mutation of exposed, hydrophobic residues in the N‐terminal helix selectively inhibits receptor, but not peptide phosphorylation, suggesting that these residues interact directly with GPCRs. Our structural and biochemical results thus provide an explanation for how receptor recognition, phospholipid binding, and kinase activation are intimately coupled in GRKs.     

G protein-coupled receptor oligomerization provides the framework for signal discrimination

The idea that G protein-coupled receptors (GPCRs) may undergo homo- or hetero-oligomerization, although highly controversial up to a few years ago, has recently gained wide acceptance. The recognition that GPCRs may exhibit either dimeric or oligomeric structures is based upon a large body of biochemical and biophysical evidence. While much effort has been spent to demonstrate the mechanism(s) by which GPCRs interact with each other, the physiological relevance of this phenomenon remains rather elusive. GPCR oligomerization has been proposed to play a role in receptor ontogeny by either chaperoning protein folding or controlling trafficking to the cell surface. However, the acquisition of these roles does not rule out the possibility that oligomeric receptors may have additional functions, once they are brought to the cell surface. Herein, we propose that protein-protein as well as protein-lipid interactions may provide the structural basis for organizing distinct cell compartments along the plasma membrane where different extracellular signals may be perceived and discriminated.     

Interaction of class A G protein-coupled receptors with G proteins

A model for interaction of classA G protein-coupled receptor with the G protein G(alpha) subunit is proposed using the rhodopsin-transducin (RD/Gt) prototype. The model combines the resolved interactions/distances, essential in the active RD*/Gt system, with the structure of Gt(alpha) C-terminal peptide bound to RD* while stabilizing it. Assuming the interactions involve conserved parts of the partners, the model specifies the conserved Helix 2 non-polar X- - -X, Helix 3 DRY and Helix 7/8 NP- -Y- - F RD* motifs interacting with the Gt(alpha) C-terminal peptide, in compliance with the structure of the latter. A concomitant role of Gt(alpha) and Gt(gamma) C-termini in stabilizing RD* could possibly be resolved assuming a receptor dimer as requisite for G protein activation.     

Multiple functions of G protein-coupled receptor kinases

Desensitization is a physiological feedback mechanism that blocks detrimental effects of persistent stimulation. G protein-coupled receptor kinase 2 (GRK2) was originally identified as the kinase that mediates G protein-coupled receptor (GPCR) desensitization. Subsequent studies revealed that GRK is a family composed of seven isoforms (GRK1–GRK7). Each GRK shows a differential expression pattern. GRK1, GRK4, and GRK7 are expressed in limited tissues. In contrast, GRK2, GRK3, GRK5, and GRK6 are ubiquitously expressed throughout the body. The roles of GRKs in GPCR desensitization are well established. When GPCRs are activated by their agonists, GRKs phosphorylate serine/threonine residues in the intracellular loops and the carboxyl-termini of GPCRs. Phosphorylation promotes translocation of β-arrestins to the receptors and inhibits further G protein activation by interrupting receptor-G protein coupling. The binding of β-arrestins to the receptors also helps to promote receptor internalization by clathrin-coated pits. Thus, the GRK-catalyzed phosphorylation and subsequent binding of β-arrestin to GPCRs are believed to be the common mechanism of GPCR desensitization and internalization. Recent studies have revealed that GRKs are also involved in the β-arrestin-mediated signaling pathway. The GRK-mediated phosphorylation of the receptors plays opposite roles in conventional G protein- and β-arrestin-mediated signaling. The GRK-catalyzed phosphorylation of the receptors results in decreased G protein-mediated signaling, but it is necessary for β-arrestin-mediated signaling. Agonists that selectively activate GRK/β-arrestin-dependent signaling without affecting G protein signaling are known as β-arrestin-biased agonists. Biased agonists are expected to have potential therapeutic benefits for various diseases due to their selective activation of favorable physiological responses or avoidance of the side effects of drugs. Furthermore, GRKs are recognized as signaling mediators that are independent of either G protein- or β-arrestin-mediated pathways. GRKs can phosphorylate non-GPCR substrates, and this is found to be involved in various physiological responses, such as cell motility, development, and inflammation. In addition to these effects, our group revealed that GRK6 expressed in macrophages mediates the removal of apoptotic cells (engulfment) in a kinase activity-dependent manner. These studies revealed that GRKs block excess stimulus and also induce cellular responses. Here, we summarized the involvement of GRKs in β-arrestin-mediated and G protein-independent signaling pathways.     

Measurement of G protein-coupled receptor surface expression

Abstract

The quantity of G protein-coupled receptors (GPCRs) expressed on the cell surface is an important factor regulating receptor signaling. Maturation, internalization, recycling and degradation together determine the net amount of receptor surface expression. Understanding every aspect of the receptor lifecycle will facilitate the development of therapeutic applications. A number of assays for measuring the surface expression of GPCRs are currently available. This minireview summarizes the currently available assays and their suitability and usage for measuring GPCR surface expression.