Identification of the orphan G protein-coupled receptor GPR31 as a receptor for 12-(S)-hydroxyeicosatetraenoic acid

Hydroxy fatty acids are critical lipid mediators involved in various pathophysiologic functions. We cloned and identified GPR31, a plasma membrane orphan G protein-coupled receptor that displays high affinity for the human 12-lipoxygenase-derived product 12-(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE). Thus, GPR31 is named 12-(S)-HETE receptor (12-HETER) in this study. The cloned 12-HETER demonstrated high affinity binding for 12-(S)-[(3)H]HETE (K(d) = 4.8 ± 0.12 nm). Also, 12-(S)-HETE efficiently and selectively stimulated GTPγS coupling in the membranes of 12-HETER-transfected cells (EC(50) = 0.28 ± 1.26 nm). Activating GTPγS coupling with 12-(S)-HETE proved to be both regio- and stereospecific. Also, 12-(S)-HETE/12-HETER interactions lead to activation of ERK1/2, MEK, and NFκB. Moreover, knocking down 12-HRTER specifically inhibited 12-(S)-HETE-stimulated cell invasion. Thus, 12-HETER represents the first identified high affinity receptor for the 12-(S)-HETE hydroxyl fatty acids.     

Functional homomers and heteromers of dopamine D2L and D3 receptors co-exist at the cell surface

Human dopamine D(2long) and D(3) receptors were modified by N-terminal addition of SNAP or CLIP forms of O(6)-alkylguanine-DNA-alkyltransferase plus a peptide epitope tag. Cells able to express each of these four constructs only upon addition of an antibiotic were established and used to confirm regulated and inducible control of expression, the specificity of SNAP and CLIP tag covalent labeling reagents, and based on homogenous time-resolved fluorescence resonance energy transfer, the presence of cell surface D(2long) and D(3) receptor homomers. Following constitutive expression of reciprocal constructs, potentially capable of forming and reporting the presence of cell surface D(2long)-D(3) heteromers, individual clones were assessed for levels of expression of the constitutively expressed protomer. This was unaffected by induction of the partner protomer and the level of expression of the partner required to generate detectable cell surface D(2long)-D(3) heteromers was defined. Such homomers and heteromers were found to co-exist and using a reconstitution of function approach both homomers and heteromers of D(2long) and D(3) receptors were shown to be functional, potentially via trans-activation of associated G protein. These studies demonstrate the ability of dopamine D(2long) and D(3) receptors to form both homomers and heteromers, and show that in cells expressing each subtype a complex mixture of homomers and heteromers co-exists at steady state. These data are of potential importance both to disorders in which D(2long) and D(3) receptors are implicated, like schizophrenia and Parkinson disease, and also to drugs exerting their actions via these sites.     

Role of SAP97 Protein in the Regulation of Corticotropin-releasing Factor Receptor 1 Endocytosis and Extracellular Signal-regulated Kinase 1/2 Signaling

The corticotropin-releasing factor (CRF) receptor 1 (CRFR1) is a target for the treatment of psychiatric diseases such as depression, schizophrenia, anxiety disorder, and bipolar disorder. The carboxyl-terminal tail of the CRFR1 terminates in a PDZ-binding motif that provides a potential site for the interaction of PSD-95/Discs Large/Zona Occludens 1 (PDZ) domain-containing proteins. In this study, we found that CRFR1 interacts with synapse-associated protein 97 (SAP97; also known as DLG1) by co-immunoprecipitation in human embryonic 293 (HEK 293) cells and cortical brain lysates and that this interaction is dependent upon an intact PDZ-binding motif at the end of the CRFR1 carboxyl-terminal tail. Similarly, we demonstrated that SAP97 is recruited to the plasma membrane in HEK 293 cells expressing CRFR1 and that mutation of the CRFR1 PDZ-binding motif results in the redistribution of SAP97 into the cytoplasm. Overexpression of SAP97 antagonized agonist-stimulated CRFR1 internalization, whereas single hairpin (shRNA) knockdown of endogenous SAP97 in HEK 293 cells resulted in increased agonist-stimulated CRFR1 endocytosis. CRFR1 was internalized as a complex with SAP97 resulting in the redistribution of SAP97 to endocytic vesicles. Overexpression or shRNA knockdown of SAP97 did not significantly affect CRFR1-mediated cAMP formation, but SAP97 knockdown did significantly attenuate CRFR1-stimulated ERK1/2 phosphorylation in a PDZ interaction-independent manner. Taken together, our studies show that SAP97 interactions with CRFR1 attenuate CRFR1 endocytosis and that SAP97 is involved in coupling G protein-coupled receptors to the activation of the ERK1/2 signaling pathway.     

Cell adhesion receptor GPR133 couples to Gs protein

Adhesion G protein-coupled receptors (GPCR), with their very large and complex N termini, are thought to participate in cell-cell and cell-matrix interactions and appear to be highly relevant in several developmental processes. Their intracellular signaling is still poorly understood. Here we demonstrate that GPR133, a member of the adhesion GPCR subfamily, activates the G(s) protein/adenylyl cyclase pathway. The presence of the N terminus and the cleavage at the GPCR proteolysis site are not required for G protein signaling. G(s) protein coupling was verified by Gα(s) knockdown with siRNA, overexpression of Gα(s), co-expression of the chimeric Gq(s4) protein that routes GPR133 activity to the phospholipase C/inositol phosphate pathway, and missense mutation within the transmembrane domain that abolished receptor activity without changing cell surface expression. It is likely that not only GPR133 but also other adhesion GPCR signal via classical receptor/G protein-interaction.     

Mapping structural determinants within third intracellular loop that direct signaling specificity of type 1 corticotropin-releasing hormone receptor

The type 1 corticotropin-releasing hormone receptor (CRH-R1) influences biological responses important for adaptation to stressful stimuli, through activation of multiple downstream effectors. The structural motifs within CRH-R1 that mediate G protein activation and signaling selectivity are unknown. The aim of this study was to gain insights about important structural determinants within the third intracellular loop (IC3) of the human CRH-R1α important for cAMP and ERK1/2 pathways activation and selectivity. We investigated the role of the juxtamembrane regions of IC3 by mutating amino acid cassettes or specific residues to alanine. Although simultaneous tandem alanine mutations of both juxtamembrane regions Arg(292)-Met(295) and Lys(311)-Lys(314) reduced ligand binding and impaired signaling, all other mutant receptors retained high affinity binding, indistinguishable from wild-type receptor. Agonist-activated receptors with tandem mutations at the proximal or distal terminal segments enhanced activation of adenylyl cyclase by 50-75% and diminished activation of inositol trisphosphate and ERK1/2 by 60-80%. Single Ala mutations identified Arg(292), Lys(297), Arg(310), Lys(311), and Lys(314) as important residues for the enhanced activation of adenylyl cyclase, partly due to reduced inhibition of adenylyl cyclase activity by pertussis toxin-sensitive G proteins. In contrast, mutation of Arg(299) reduced receptor signaling activity and cAMP response. Basic as well as aliphatic amino acids within both juxtamembrane regions were identified as important for ERK1/2 phosphorylation through activation of pertussis toxin-sensitive G proteins as well as G(q) proteins. These data uncovered unexpected roles for key amino acids within the highly conserved hydrophobic N- and C-terminal microdomains of IC3 in the coordination of CRH-R1 signaling activity.     

The N terminus of the adhesion G protein-coupled receptor GPR56 controls receptor signaling activity

GPR56 is an adhesion G protein-coupled receptor that plays a key role in cortical development. Mutations to GPR56 in humans cause malformations of the cerebral cortex, but little is known about the normal function of the receptor. We found that the large N terminus (NT) of GPR56 is cleaved from the rest of the receptor during processing but remains non-covalently associated with the seven-transmembrane region of the receptor, as indicated by coimmunoprecipitation of the two GPR56 fragments from both transfected cells and native tissue. We also found that truncation of the GPR56 NT results in constitutive activation of receptor signaling, as revealed by increased GPR56-stimulated signaling upon transfection of HEK-293 cells with truncated GPR56, greatly enhanced binding of β-arrestins by truncated GPR56 relative to the full-length receptor, extensive ubiquitination of truncated GPR56, and cytotoxicity induced by truncated GPR56 that could be rescued by cotransfection of cells with β-arrestin 2. Furthermore, we found that the GPR56 NT is capable of homophilic trans-trans interactions that enhance receptor signaling activity. On the basis of these findings, we suggest a model of receptor activation in which the large N terminus of GPR56 constrains receptor activity but N-terminal interactions (GPR56 NT with an extracellular ligand and/or GPR56 NT homophilic trans-trans associations) can remove this inhibitory influence of the N terminus to activate receptor signaling.     

Calcyon Forms a Novel Ternary Complex with Dopamine D1 Receptor through PSD-95 Protein and Plays a Role in Dopamine Receptor Internalization

Calcyon, once known for interacting directly with the dopamine D(1) receptor (D(1)DR), is implicated in various neuropsychiatric disorders including schizophrenia, bipolar disorder, and attention deficit hyperactivity disorder. Although its direct interaction with D(1)DR has been shown to be misinterpreted, it still plays important roles in D(1)DR signaling. Here, we found that calcyon interacts with the PSD-95 and subsequently forms a ternary complex with D(1)DR through PSD-95. Calcyon is phosphorylated on Ser-169 by the PKC activator phorbol 12-myristate 13-acetate or by the D(1)DR agonist SKF-81297, and its phosphorylation increases its association with PSD-95 and recruitment to the cell surface. Interestingly, the internalization of D(1)DR at the cell surface was enhanced by phorbol 12-myristate 13-acetate and SKF-81297 in the presence of calcyon, but not in the presence of its S169A phospho-deficient mutant, suggesting that the phosphorylation of calcyon and the internalization of the surface D(1)DR are tightly correlated. Our results suggest that calcyon regulates D(1)DR trafficking by forming a ternary complex with D(1)DR through PSD-95 and thus possibly linking glutamatergic and dopamine receptor signalings. This also raises the possibility that a novel ternary complex could represent a potential therapeutic target for the modulation of related neuropsychiatric disorders.     

Neurotensin triggers dopamine D2 receptor desensitization through a protein kinase C and beta-arrestin1-dependent mechanism

The peptide neurotensin (NT) is known to exert a potent excitatory effect on the dopaminergic system by inhibiting D2 dopamine (DA) receptor (D2R) function. This regulation is dependent on activation of PKC, a well known effector of the type 1 NT receptor (NTR1). Because PKC phosphorylation of the D2R has recently been shown to induce its internalization, we hypothesized that NT acts to reduce D2R function through heterologous desensitization of the D2R. In the present study, we first used HEK-293 cells to demonstrate that NT induces PKC-dependent D2R internalization. Furthermore, internalization displayed faster kinetics in cells expressing the D2R short isoform, known to act as an autoreceptor in DA neurons, than in cells expressing the long isoform, known to act as a postsynaptic D2R. In patch clamp experiments on cultured DA neurons, overexpression of a mutant D2S lacking three key PKC phosphorylation sites abrogated the ability of NT to reduce D2R-mediated cell firing inhibition. Short interfering RNA-mediated inhibition of β-arrestin1 and dynamin2, proteins important for receptor desensitization, reduced agonist-induced desensitization of D2R function, but only the inhibition of β-arrestin1 reduced the effect of NT on D2R function. Taken together, our data suggest that NT acutely regulates D2 autoreceptor function and DA neuron excitability through PKC-mediated phosphorylation of the D2R, leading to heterologous receptor desensitization.     

Altered immune response in mice deficient for the G protein-coupled receptor GPR34

The X-chromosomal GPR34 gene encodes an orphan G(i) protein-coupled receptor that is highly conserved among vertebrates. To evaluate the physiological relevance of GPR34, we generated a GPR34-deficient mouse line. GPR34-deficient mice were vital, reproduced normally, and showed no gross abnormalities in anatomical, histological, laboratory chemistry, or behavioral investigations under standard housing. Because GPR34 is highly expressed in mononuclear cells of the immune system, mice were specifically tested for altered functions of these cell types. Following immunization with methylated BSA, the number of granulocytes and macrophages in spleens was significantly lower in GPR34-deficient mice as in wild-type mice. GPR34-deficient mice showed significantly increased paw swelling in the delayed type hypersensitivity test and higher pathogen burden in extrapulmonary tissues after pulmonary infection with Cryptococcus neoformans compared with wild-type mice. The findings in delayed type hypersensitivity and infection tests were accompanied by significantly different basal and stimulated TNF-α, GM-CSF, and IFN-γ levels in GPR34-deficient animals. Our data point toward a functional role of GPR34 in the cellular response to immunological challenges.     

STR-33, a novel G protein-coupled receptor that regulates locomotion and egg laying in Caenorhabditis elegans

Despite their predicted functional importance, most G protein-coupled receptors (GPCRs) in Caenorhabditis elegans have remained largely uncharacterized. Here, we focused on one GPCR, STR-33, encoded by the str-33 gene, which was discovered through a ligand-based screening procedure. To characterize STR-33 function, we performed UV-trimethylpsolaren mutagenesis and isolated an str-33-null mutant. The resulting mutant showed hypersinusoidal movement and a hyperactive egg-laying phenotype. Two types of egg laying-related mutations have been characterized: egg laying-deficient (Egl-d) and hyperactive egg laying (Egl-c). The defect responsible for the egg laying-deficient Egl-d phenotype is related to Gα(q) signaling, whereas that responsible for the opposite, hyperactive egg-laying Egl-c phenotype is related to Gα(o) signaling. We found that the hyperactive egg-laying defect of the str-33(ykp001) mutant is dependent on the G protein GOA-1/Gα(o). Endogenous acetylcholine suppressed egg laying in C. elegans via a Gα(o)-signaling pathway by inhibiting serotonin biosynthesis or release from the hermaphrodite-specific neuron. Consistent with this, in vivo expression of the serotonin biosynthetic enzyme, TPH-1, was up-regulated in the str-33(ykp001) mutant. Taken together, these results suggest that the GPCR, STR-33, may be one of the neurotransmitter receptors that regulates locomotion and egg laying in C. elegans.     

Calcium-sensing receptor decreases cell surface expression of the inwardly rectifying K+ channel Kir4.1

The Ca(2+)-sensing receptor (CaR) regulates salt and water transport in the kidney as demonstrated by the association of gain of function CaR mutations with a Bartter syndrome-like, salt-wasting phenotype, but the precise mechanism for this effect is not fully established. We found previously that the CaR interacts with and inactivates an inwardly rectifying K(+) channel, Kir4.1, which is expressed in the distal nephron that contributes to the basolateral K(+) conductance, and in which loss of function mutations are associated with a complex phenotype that includes renal salt wasting. We now find that CaR inactivates Kir4.1 by reducing its cell surface expression. Mutant CaRs reduced Kir4.1 cell surface expression and current density in HEK-293 cells in proportion to their signaling activity. Mutant, activated Gα(q) reduced cell surface expression and current density of Kir4.1, and these effects were blocked by RGS4, a protein that blocks signaling via Gα(i) and Gα(q). Other α subunits had insignificant effects. Knockdown of caveolin-1 blocked the effect of Gα(q) on Kir4.1, whereas knockdown of the clathrin heavy chain had no effect. CaR had no comparable effect on the renal outer medullary K(+) channel, an apical membrane distal nephron K(+) channel that is internalized by clathrin-coated vesicles. Co-immunoprecipitation studies showed that the CaR and Kir4.1 physically associate with caveolin-1 in HEK cells and in kidney extracts. Thus, the CaR decreases cell surface expression of Kir4.1 channels via a mechanism that involves Gα(q) and caveolin. These results provide a novel molecular basis for the inhibition of renal NaCl transport by the CaR.     

Dopamine D(3) receptors are down-regulated following heterologous endocytosis by a specific interaction with G protein-coupled receptor-associated sorting protein-1

The D(3) dopamine receptor is endocytosed through a heterologous mechanism mediated by phorbol esters. Here, we show that following this endocytosis the D(3) dopamine receptors fail to recycle and are instead targeted for degradation through an interaction with the G protein-coupled receptor (GPCR)-associated sorting protein-1 (GASP-1). Furthermore, we identified a specific binding motif in the C terminus common to the D(3) and D(2) that confers GASP-1 binding. shRNA knockdown of GASP-1 delayed post-endocytic degradation of both the D(2) and D(3) dopamine receptors. In addition, mutation of the D(2) and D(3) receptor C termini to resemble the D(4), which does not interact with GASP-1, not only inhibited GASP-1 binding but slowed degradation after endocytosis. Conversely, mutation of the C terminus of the D(4) to resemble that of the D(2) and D(3) facilitated GASP-1 binding and promoted post-endocytic degradation of the mutant D(4) receptor. Thus, we have identified a motif that is both necessary and sufficient to promote GASP-1 binding and receptor degradation. In addition, these data demonstrated that GASP-1 can mediate post-endocytic degradation of dopamine receptors that have been endocytosed not only as a consequence of dopamine activation but also as a consequence of activation by phorbol esters.     

Roles of phosphorylation-dependent and -independent mechanisms in the regulation of histamine H2 receptor by G protein-coupled receptor kinase 2

It is widely assumed that G protein-coupled receptor kinase 2 (GRK2)-mediated specific inhibition of G protein-coupled receptors (GPCRs) response involves GRK-mediated receptor phosphorylation followed by β-arrestin binding and subsequent uncoupling from the heterotrimeric G protein. It has recently become evident that GRK2-mediated GPCRs regulation also involves phosphorylation-independent mechanisms. In the present study we investigated whether the histamine H2 receptor (H2R), a Gα(s)-coupled GPCR known to be desensitized by GRK2, needs to be phosphorylated for its desensitization and/or internalization and resensitization. For this purpose we evaluated the effect of the phosphorylating-deficient GRK2K220R mutant on H2R signaling in U937, COS7, and HEK293T cells. We found that although this mutant functioned as dominant negative concerning receptor internalization and resensitization, it desensitized H2R signaling in the same degree as the GRK2 wild type. To identify the domains responsible for the kinase-independent receptor desensitization, we co-transfected the receptor with constructions encoding the GRK2 RGS-homology domain (RH) and the RH or the kinase domain fused to the pleckstrin-homology domain. Results demonstrated that the RH domain of GRK2 was sufficient to desensitize the H2R. Moreover, disruption of RGS functions by the use of GRK2D110A/K220R double mutant, although coimmunoprecipitating with the H2R, reversed GRK2K220R-mediated H2R desensitization. Overall, these results indicate that GRK2 induces desensitization of H2R through a phosphorylation-independent and RGS-dependent mechanism and extends the GRK2 RH domain-mediated regulation of GPCRs beyond Gα(q)-coupled receptors. On the other hand, GRK2 kinase activity proved to be necessary for receptor internalization and the resulting resensitization.     

Sphingosine 1-Phosphate Receptor 4 Uses HER2 (ERBB2) to Regulate Extracellular Signal Regulated Kinase-1/2 in MDA-MB-453 Breast Cancer Cells

We demonstrate here that the bioactive lipid sphingosine 1-phosphate (S1P) uses sphingosine 1-phosphate receptor 4 (S1P₄) and human epidermal growth factor receptor 2 (HER2) to stimulate the extracellular signal regulated protein kinase 1/2 (ERK-1/2) pathway in MDA-MB-453 cells. This was based on several lines of evidence. First, the S1P stimulation of ERK-1/2 was abolished by JTE013, which we show here is an S1P_2/4 antagonist and reduced by siRNA knockdown of S1P₄. Second, the S1P-stimulated activation of ERK-1/2 was almost completely abolished by a HER2 inhibitor (ErbB2 inhibitor II) and reduced by siRNA knockdown of HER2 expression. Third, phyto-S1P, which is an S1P₄ agonist, stimulated ERK-1/2 activation in an S1P₄- and HER2-dependent manner. Fourth, FTY720 phosphate, which is an agonist at S1P_1,3,4,5 but not S1P₂ stimulated activation of ERK-1/2. Fifth, S1P stimulated the tyrosine phosphorylation of HER2, which was reduced by JTE013. HER2 which is an orphan receptor tyrosine kinase is the preferred dimerization partner of the EGF receptor. However, EGF-stimulated activation of ERK-1/2 was not affected by siRNA knockdown of HER2 or by ErbB2 (epidermal growth factor receptor 2 (or HER2)) inhibitor II in MDA-MB-453 cells. Moreover, S1P-stimulated activation of ERK-1/2 does not require an EGF receptor. Thus, S1P and EGF function in a mutually exclusive manner. In conclusion, the magnitude of the signaling gain on the ERK-1/2 pathway produced in response to S1P can be increased by HER2 in MDA-MB-453 cells. The linkage of S1P with an oncogene suggests that S1P and specifically S1P₄ may have an important role in breast cancer progression.     

Role of the transmembrane domain 4/extracellular loop 2 junction of the human gonadotropin-releasing hormone receptor in ligand binding and receptor conformational selection

Recent crystal structures of G protein-coupled receptors (GPCRs) show the remarkable structural diversity of extracellular loop 2 (ECL2), implying its potential role in ligand binding and ligand-induced receptor conformational selectivity. Here we have applied molecular modeling and mutagenesis studies to the TM4/ECL2 junction (residues Pro(174(4.59))-Met(180(4.66))) of the human gonadotropin-releasing hormone (GnRH) receptor, which uniquely has one functional type of receptor but two endogenous ligands in humans. We suggest that the above residues assume an α-helical extension of TM4 in which the side chains of Gln(174(4.60)) and Phe(178(4.64)) face toward the central ligand binding pocket to make H-bond and aromatic contacts with pGlu(1) and Trp(3) of both GnRH I and GnRH II, respectively. The interaction between the side chains of Phe(178(4.64)) of the receptor and Trp(3) of the GnRHs was supported by reciprocal mutations of the interacting residues. Interestingly, alanine mutations of Leu(175(4.61)), Ile(177(4.63)), and Met(180(4.66)) decreased mutant receptor affinity for GnRH I but, in contrast, increased affinity for GnRH II. This suggests that these residues make intramolecular or intermolecular contacts with residues of transmembrane (TM) domain 3, TM5, or the phospholipid bilayer, which couple the ligand structure to specific receptor conformational switches. The marked decrease in signaling efficacy of I177A and F178A also indicates that IIe(177(4.63)) and Phe(178(4.64)) are important in stabilizing receptor-active conformations. These findings suggest that the TM4/ECL2 junction is crucial for peptide ligand binding and, consequently, for ligand-induced receptor conformational selection.     

Involvement of protein kinase Cdelta/extracellular signal-regulated kinase/poly(ADP-ribose) polymerase-1 (PARP-1) signaling pathway in histamine-induced up-regulation of histamine H1 receptor gene expression in HeLa cells

The histamine H(1) receptor (H1R) gene is up-regulated in patients with allergic rhinitis. However, the mechanism and reason underlying this up-regulation are still unknown. Recently, we reported that the H1R expression level is strongly correlated with the severity of allergic symptoms. Therefore, understanding the mechanism of this up-regulation will help to develop new anti-allergic drugs targeted for H1R gene expression. Here we studied the molecular mechanism of H1R up-regulation in HeLa cells that express H1R endogenously in response to histamine and phorbol 12-myristate 13-acetate (PMA). In HeLa cells, histamine stimulation caused up-regulation of H1R gene expression. Rottlerin, a PKCδ-selective inhibitor, inhibited up-regulation of H1R gene expression, but Go6976, an inhibitor of Ca(2+)-dependent PKCs, did not. Histamine or PMA stimulation resulted in PKCδ phosphorylation at Tyr(311) and Thr(505). Activation of PKCδ by H(2)O(2) resulted in H1R mRNA up-regulation. Overexpression of PKCδ enhanced up-regulation of H1R gene expression, and knockdown of the PKCδ gene suppressed this up-regulation. Histamine or PMA caused translocation PKCδ from the cytosol to the Golgi. U0126, an MEK inhibitor, and DPQ, a poly(ADP-ribose) polymerase-1 inhibitor, suppressed PMA-induced up-regulation of H1R gene expression. These results were confirmed by a luciferase assay using the H1R promoter. Phosphorylation of ERK and Raf-1 in response to PMA was also observed. However, real-time PCR analysis showed no inhibition of H1R mRNA up-regulation by a Raf-1 inhibitor. These results suggest the involvement of the PKCδ/ERK/poly(ADP-ribose) polymerase-1 signaling pathway in histamine- or PMA-induced up-regulation of H1R gene expression in HeLa cells.     

Second extracellular loop of human glucagon-like peptide-1 receptor (GLP-1R) has a critical role in GLP-1 peptide binding and receptor activation

The glucagon-like peptide-1 receptor (GLP-1R) is a therapeutically important family B G protein-coupled receptor (GPCR) that is pleiotropically coupled to multiple signaling effectors and, with actions including regulation of insulin biosynthesis and secretion, is one of the key targets in the management of type II diabetes mellitus. However, there is limited understanding of the role of the receptor core in orthosteric ligand binding and biological activity. To assess involvement of the extracellular loop (ECL) 2 in ligand-receptor interactions and receptor activation, we performed alanine scanning mutagenesis of loop residues and assessed the impact on receptor expression and GLP-1(1-36)-NH(2) or GLP-1(7-36)-NH(2) binding and activation of three physiologically relevant signaling pathways as follows: cAMP formation, intracellular Ca(2+) (Ca(2+)(i)) mobilization, and phosphorylation of extracellular signal-regulated kinases 1 and 2 (pERK1/2). Although antagonist peptide binding was unaltered, almost all mutations affected GLP-1 peptide agonist binding and/or coupling efficacy, indicating an important role in receptor activation. However, mutation of several residues displayed distinct pathway responses with respect to wild type receptor, including Arg-299 and Tyr-305, where mutation significantly enhanced both GLP-1(1-36)-NH(2)- and GLP-1(7-36)-NH(2)-mediated signaling bias for pERK1/2. In addition, mutation of Cys-296, Trp-297, Asn-300, Asn-302, and Leu-307 significantly increased GLP-1(7-36)-NH(2)-mediated signaling bias toward pERK1/2. Of all mutants studied, only mutation of Trp-306 to alanine abolished all biological activity. These data suggest a critical role of ECL2 of the GLP-1R in the activation transition(s) of the receptor and the importance of this region in the determination of both GLP-1 peptide- and pathway-specific effects.     

Macromolecular Composition Dictates Receptor and G Protein Selectivity of Regulator of G Protein Signaling (RGS) 7 and 9-2 Protein Complexes in Living Cells

Regulator of G protein signaling (RGS) proteins play essential roles in the regulation of signaling via G protein-coupled receptors (GPCRs). With hundreds of GPCRs and dozens of G proteins, it is important to understand how RGS regulates selective GPCR-G protein signaling. In neurons of the striatum, two RGS proteins, RGS7 and RGS9-2, regulate signaling by μ-opioid receptor (MOR) and dopamine D2 receptor (D2R) and are implicated in drug addiction, movement disorders, and nociception. Both proteins form trimeric complexes with the atypical G protein β subunit Gβ5 and a membrane anchor, R7BP. In this study, we examined GTPase-accelerating protein (GAP) activity as well as Gα and GPCR selectivity of RGS7 and RGS9-2 complexes in live cells using a bioluminescence resonance energy transfer-based assay that monitors dissociation of G protein subunits. We showed that RGS9-2/Gβ5 regulated both Gi and Go with a bias toward Go, but RGS7/Gβ5 could serve as a GAP only for Go. Interestingly, R7BP enhanced GAP activity of RGS7 and RGS9-2 toward Go and Gi and enabled RGS7 to regulate Gi signaling. Neither RGS7 nor RGS9-2 had any activity toward Gz, Gs, or Gq in the absence or presence of R7BP. We also observed no effect of GPCRs (MOR and D2R) on the G protein bias of R7 RGS proteins. However, the GAP activity of RGS9-2 showed a strong receptor preference for D2R over MOR. Finally, RGS7 displayed an four times greater GAP activity relative to RGS9-2. These findings illustrate the principles involved in establishing G protein and GPCR selectivity of striatal RGS proteins.     

Dopamine D2 Receptor Relies upon PPM/PP2C Protein Phosphatases to Dephosphorylate Huntingtin Protein

Striatal dopamine D2 receptor (D2R) relies upon G protein- and β-arrestin-dependent signaling pathways to convey its action on motor control and behavior. Considering that D2R activation inhibits Akt in the striatum and that huntingtin physiological functions are affected by Akt phosphorylation, we sought to investigate whether D2R-mediated signaling could regulate huntingtin phosphorylation. We demonstrate that D2R activation decreases huntingtin phosphorylation on its Akt site. This dephosphorylation event depends upon the Gα_i-dependent engagement of specific members of the protein phosphatase metallo-dependent (PPM/PP2C) family and is independent of β-arrestin 2. These observations identify the PPM/PP2C family as a mediator of G protein-coupled receptor signaling and thereby suggest a novel mechanism of dopaminergic signaling.