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.     

Biologically active peptides interacting with the G protein-coupled formylpeptide receptor

Leucocytes accumulate at sites of inflammation and microbial infection in response to locally produced chemotactic factors. N-formylpeptides produced by Gram negative bacteria were among the first chemotactic factors structurally defined which signal through G protein-coupled formylpeptide receptor (FPR) and FPR-like 1 (FPRL1) expressed by phagocytic leukocytes in human and in mouse homogogues mFPR and mFPR2. During the past few years, a number of pathogen- and host-derived agonists/antagonists for FPR, FPRL1 and another FPR variant FPR-like 2 (FPRL2) have been identified. Activation of formylpeptide receptors (FPRs) in phagocytic leukocytes by agonists results in increased cell chemotaxis, phagocytosis, and release of pro-inflammatory mediators. Peptide agonists for FPRs have also been shown to possess immune adjuvant activity when injected in mice. In addition, FPR aberrantly expressed on highly malignant human glioblastoma cells promotes tumor cell migration, proliferation and production of vascular endothelial growth factor in response to agonists released by necrotic tumor cells. Therefore, formylpeptide receptor ligands, by interacting with FPRs, play important roles in host defense and in the rapid progression of human glioblastoma.     

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 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.     

Cloning and characterization of additional members of the G protein-coupled receptor family

A search of the expressed sequence tag (EST) database retrieved a human cDNA sequence which partially encoded a novel G protein-coupled receptor (GPCR) GPR26. A human genomic DNA fragment encoding a partial open reading frame (ORF) and a rat cDNA encoding the full length ORF of GPR26 were obtained by library screening. The rat GPR26 cDNA encoded a protein of 317 amino acids, most similar (albeit distantly related) to the serotonin 5-HT(5A) and gastrin releasing hormone BB2 receptors. GPR26 mRNA expression analysis revealed signals in the striatum, pons, cerebellum and cortex. HEK293 and Rh7777 cells transfected with GPR26 cDNA displayed high basal cAMP levels, slow growth rate of clonal populations and derangements of normal cell shape. We also used a sequence reported only in the patent literature encoding GPR57 (a.k.a. HNHCI32) to PCR amplify a DNA fragment which was used to screen a human genomic library. This resulted in the cloning of a genomic fragment containing a pseudogene, psiGPR57, with a 99.6% nucleotide identity to GPR57. Based on shared sequence identities, the receptor encoded by GPR57 was predicted to belong to a novel subfamily of GPCRs together with GPR58 (a.k.a. phBL5, reported only in the patent literature), putative neurotransmitter receptor (PNR) and a 5-HT(4) pseudogene. Analysis of this subfamily revealed greatest identities (approximately 56%) between the receptors encoded by GPR57 and GPR58, each with shared identities of approximately 40% with PNR. Furthermore, psiGPR57, GPR58, PNR and the 5-HT(4) pseudogene were mapped in a cluster localized to chromosome 6q22-24. PNR and GPR58 were expressed in COS cells, however no specific binding was observed for various serotonin receptor-specific ligands.     

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.     

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.     

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.     

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.     

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.     

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.     

Clathrin-dependent mechanisms of G protein-coupled receptor endocytosis

The heptahelical G protein-coupled receptor (GPCR) family includes approximately 900 members and is the largest family of signaling receptors encoded in the mammalian genome. G protein-coupled receptors elicit cellular responses to diverse extracellular stimuli at the plasma membrane and some internalized receptors continue to signal from intracellular compartments. In addition to rapid desensitization, receptor trafficking is critical for regulation of the temporal and spatial aspects of GPCR signaling. Indeed, GPCR internalization functions to control signal termination and propagation as well as receptor resensitization. Our knowledge of the mechanisms that regulate mammalian GPCR endocytosis is based predominantly on arrestin regulation of receptors through a clathrin- and dynamin-dependent pathway. However, multiple clathrin adaptors, which recognize distinct endocytic signals, are now known to function in clathrin-mediated endocytosis of diverse cargo. Given the vast number and diversity of GPCRs, the complexity of clathrin-mediated endocytosis and the discovery of multiple clathrin adaptors, a single universal mechanism controlling endocytosis of all mammalian GPCRs is unlikely. Indeed, several recent studies now suggest that endocytosis of different GPCRs is regulated by distinct mechanisms and clathrin adaptors. In this review, we discuss the diverse mechanisms that regulate clathrin-dependent GPCR endocytosis.     

Regulation of G protein-coupled receptor export trafficking

G protein-coupled receptors (GPCRs) constitute a superfamily of cell-surface receptors which share a common topology of seven transmembrane domains and modulate a variety of cell functions through coupling to heterotrimeric G proteins by responding to a vast array of stimuli. The magnitude of cellular response elicited by a given signal is dictated by the level of GPCR expression at the plasma membrane, which is the balance of elaborately regulated endocytic and exocytic trafficking. This review will cover recent advances in understanding the molecular mechanism underlying anterograde transport of the newly synthesized GPCRs from the endoplasmic reticulum (ER) through the Golgi to the plasma membrane. We will focus on recently identified motifs involved in GPCR exit from the ER and the Golgi, GPCR folding in the ER and the rescue of misfolded receptors from within, GPCR-interacting proteins that modulate receptor cell-surface targeting, pathways that mediate GPCR traffic, and the functional role of export in controlling GPCR signaling.     

Regulation of G protein-coupled receptor export trafficking

G protein-coupled receptors (GPCRs) constitute a superfamily of cell-surface receptors which share a common topology of seven transmembrane domains and modulate a variety of cell functions through coupling to heterotrimeric G proteins by responding to a vast array of stimuli. The magnitude of cellular response elicited by a given signal is dictated by the level of GPCR expression at the plasma membrane, which is the balance of elaborately regulated endocytic and exocytic trafficking. This review will cover recent advances in understanding the molecular mechanism underlying anterograde transport of the newly synthesized GPCRs from the endoplasmic reticulum (ER) through the Golgi to the plasma membrane. We will focus on recently identified motifs involved in GPCR exit from the ER and the Golgi, GPCR folding in the ER and the rescue of misfolded receptors from within, GPCR-interacting proteins that modulate receptor cell-surface targeting, pathways that mediate GPCR traffic, and the functional role of export in controlling GPCR signaling.     

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.