Molecular mechanisms of G protein-coupled receptor signaling: role of G protein-coupled receptor kinases and arrestins in receptor desensitization and resensitization

Dynamic regulation of G protein-coupled receptor signaling demands a coordinated balance between mechanisms leading to the generation, turning off and re-establishment of agonist-mediated signals. G protein-coupled receptor kinases (GRKs) and arrestin proteins not only mediate agonist-dependent G protein-coupled receptor desensitization, but also initiate the internalization (sequestration) of activated receptors, a process leading to receptor resensitization. Studies on the specificity of beta-arrestin functions reveal a multiplicity of G protein-coupled receptor endocytic pathways and suggest that beta-arrestins might serve as adaptors specifically targeting receptors for dynamin-dependent clathrin-mediated endocytosis. Moreover, inactivation of the GRK2 gene in mice has lead to the discovery of an unexpected role of GRK2 in cardiac development, further emphasizing the pleiotropic function of GRKs and arrestins.     

Factors to consider in the naming of a G protein-coupled receptor subtype

Receptors that mediate their effects through G proteins are predicted to have a seven transmembrane domain architecture. The last few years have seen a remarkable increase in the cloning of members of this superfamily leading to the identification of many more receptors than previously thought to exist on the basis of differences in agonist and antagonist specificities. This has important implications for nomenclature and classification, especially in view of the difficulty in relating receptors identified through cloning techniques to endogenously expressed receptors. Receptor cloning has also identified important differences in receptors between species raising the question as to whether these should be considered as species homologues or distinct subtypes. It is also becoming increasingly apparent that the pharmacology of this superfamily of receptors is influenced by the nature of the G protein present in the host cell and by alternative splicing of the receptor. The rapid pace of developments in this area necessitate the need for a regular publication summarizing recent developments. In the future, the cloning of G protein-coupled receptors will enable rationalization of the naming of individual receptor subtypes and identification of their interrelationships.     

Molecular mechanisms of G protein-coupled receptor desensitization and resensitization

Beta-arrestin proteins play a dual role in regulating G protein-coupled receptor (GPCR) responsiveness by contributing to both receptor desensitization and internalization. Recently, beta-arrestins were also shown to be critical determinants for beta2-adrenergic receptor (beta2AR) resensitization. This was demonstrated by overexpressing wild-type beta-arrestins to rescue the resensitization-defect of a beta2AR (Y326A) mutant (gain of function) and overexpressing a dominant-negative beta-arrestin inhibitor of beta2AR sequestration to impair beta2AR dephosphorylation and resensitization (loss of function). Moreover, the ability of the beta2AR to resensitize in different cell types was shown to be dependent upon beta-arrestin expression levels. To further study the mechanisms underlying beta-arrestin function, green fluorescent protein was coupled to beta-arrestin2 (beta arr2GFP), thus allowing the real-time visualization of the agonist-dependent trafficking of beta-arrestin in living cells. Beta arr2GFP translocation from the cytoplasm to the plasma membrane proceeded with a time course, sensitivity and specificity that was indistinguishable from the most sensitive second messenger readout systems. Beta arr2GFP translocation was GRK-dependent and was demonstrated for 16 different ligand-activated GPCRs. Because beta-arrestin binding is a common divergent step in GPCR signalling, this assay represents a universal methodology for screening orphan receptors, GRK inhibitors and novel GPCR ligands. Moreover, beta arr2GFP provides a valuable new tool to dissect the biological function and regulation of beta-arrestin proteins.     

Phosphorylation and localization of Kss1, a MAP kinase of the Saccharomyces cerevisiae pheromone response pathway

Kss1 protein kinase, and the homologous Fus3 kinase, are required for pheromone signal transduction in Saccharomyces cerevisiae. In MATa haploids exposed to alpha-factor, Kss1 was rapidly phosphorylated on both Thr183 and Tyr185, and both sites were required for Kss1 function in vivo. De novo protein synthesis was required for sustained pheromone-induced phosphorylation of Kss1. Catalytically inactive Kss1 mutants displayed alpha-factor-induced phosphorylation on both residues, even in kss1 delta cells; hence, autophosphorylation is not obligatory for these modifications. In kss1 delta fus3 delta double mutants, Kss1 phosphorylation was elevated even in the absence of pheromone; thus, cross-phosphorylation by Fus3 is not responsible for Kss1 activation. In contrast, pheromone-induced Kss1 phosphorylation was eliminated in mutants deficient in two other protein kinases, Ste11 and Ste7. A dominant hyperactive allele of STE11 caused a dramatic increase in the phosphorylation of Kss1, even in the absence of pheromone stimulation, but required Ste7 for this effect, suggesting an order of function: Ste11-->Ste7-->Kss1. When overproduced, Kss1 stimulated recovery from pheromone-imposed G1 arrest. Catalytic activity was essential for Kss1 function in signal transmission, but not for its recovery-promoting activity. Kss1 was found almost exclusively in the particulate material and its subcellular fractionation was unaffected by pheromone treatment. Indirect immunofluorescence demonstrated that Kss1 is concentrated in the nucleus and that its distribution is not altered detectably during signaling.     

Binding and phosphorylation of tubulin by G protein-coupled receptor kinases

Although the beta-adrenergic receptor kinase (betaARK) mediates agonist-dependent phosphorylation and desensitization of G protein-coupled receptors, recent studies suggest additional cellular functions. During our attempts to identify novel betaARK interacting proteins, we found that the cytoskeletal protein tubulin could specifically bind to a betaARK-coupled affinity column. In vitro analysis demonstrated that betaARK and G protein-coupled receptor kinase-5 (GRK5) were able to stoichiometrically phosphorylate purified tubulin dimers with a preference for beta-tubulin and, under certain conditions, the betaIII-isotype. Examination of the GRK/tubulin binding characteristics revealed that tubulin dimers and assembled microtubules bind GRKs, whereas the catalytic domain of betaARK contains the primary tubulin binding determinants. In vivo interaction of GRK and tubulin was suggested by the following: (i) co-purification of betaARK with tubulin from brain tissue; (ii) co-immunoprecipitation of betaARK and tubulin from COS-1 cells; and (iii) co-localization of betaARK and GRK5 with microtubule structures in COS-1 cells. In addition, GRK-phosphorylated tubulin was found preferentially associated with the microtubule fraction during in vitro assembly assays suggesting potential functional significance. These results suggest a novel link between the cytoskeleton and GRKs that may be important for regulating GRK and/or tubulin function.     

Transgenic manipulation of myocardial G protein-coupled receptors and receptor kinases

The rare folate-sensitive, fragile sites on chromsomes X, 11, and 16 contain blocks of CCG triplet repeats and large expansions of the CCG block at the FRAXA site produce the fragile X syndrome (FraX). The fragile, poorly staining nature of these sites suggested an altered chromatin structure. Here, repeating CCG DNAs from FraX patients were tested for their ability to assemble into nucleosomes, the basic subunits of chromatin, using in vitro nucleosome reconstitution, electron microscopy and competitive assembly gel retardation assays. CCG blocks of >50 repeats displayed strong nucleosome exclusion, providing a possible explanation for the nature of these sites.     

Sphingosine-1-phosphate as a ligand for the G protein-coupled receptor EDG-1

The sphingolipid metabolite sphingosine-1-phosphate (SPP) has been implicated as a second messenger in cell proliferation and survival. However, many of its biological effects are due to binding to unidentified receptors on the cell surface. SPP activated the heterotrimeric guanine nucleotide binding protein (G protein)-coupled orphan receptor EDG-1, originally cloned as Endothelial Differentiation Gene-1. EDG-1 bound SPP with high affinity (dissociation constant = 8.1 nM) and high specificity. Overexpression of EDG-1 induced exaggerated cell-cell aggregation, enhanced expression of cadherins, and formation of well-developed adherens junctions in a manner dependent on SPP and the small guanine nucleotide binding protein Rho.     

Substitutions in the pheromone-responsive Gbeta protein of Saccharomyces cerevisiae confer a defect in recovery from pheromone treatment

The pheromone-responsive Galpha protein of Saccharomyces cerevisiae, Gpa1p, stimulates an adaptive mechanism that downregulates the mating signal. In a genetic screen designed to identify signaling elements required for Gpa1p-mediated adaptation, a large collection of adaptive-defective (Adp-) mutants were recovered. Of the 49 mutants characterized thus far, approximately three-quarters exhibit a dominant defect in the negative regulation of the pheromone response. Eight of the dominant Adp- mutations showed tight linkage to the gene encoding the pheromone-responsive Gbeta, STE4. Sequence analysis of the STE4 locus in the relevant mutant strains revealed seven novel STE4 alleles, each of which was shown to disrupt proper regulation of the pheromone response. Although the STE4 mutations had only minor effects on basal mating pathway activity, the mutant forms of Gbeta dramatically affected the ability of the cell to turn off the mating response after exposure to pheromone. Moreover, the signaling activity of the aberrant Gbetagamma subunits was suppressed by G322E, a mutant form of Gpa1p that blocks the pheromone response by sequestering Gbetagamma, but not by E364K, a hyperadaptive form of Gpa1p. On the basis of these observations, we propose that Gpa1p-mediated adaptation involves the binding of an unknown negative regulator to Gbetagamma.     

Desensitization of the delta-opioid receptor correlates with its phosphorylation in SK-N-BE cells: involvement of a G protein-coupled receptor kinase

Phosphorylation of G protein-coupled receptors is considered an important step during their desensitization. In SK-N-BE cells, recently presented as a pertinent model for the studies of the human delta-opioid receptor, pretreatment with the opioid agonist etorphine increased time-dependently the rate of phosphorylation of a 51-kDa membrane protein. Immunological characterization of this protein with an antibody, raised against the amino-terminal region of the cloned human delta-opioid receptor, revealed that it corresponded to the delta-opioid receptor. During prolonged treatment with etorphine, phosphorylation increased as early as 15 min to reach a maximum within 1 h. Phosphorylation and desensitization of adenylyl cyclase inhibition paralleled closely and okadaic acid inhibited the resensitization, a result strongly suggesting that phosphorylation of the delta-opioid receptor plays a prominent role in its rapid desensitization. The increase in phosphorylation of the delta-opioid receptor, as well as its desensitization, was not affected by H7, an inhibitor of protein kinase A and protein kinase C, but was drastically reduced by heparin or Zn2+, known to act as G protein-coupled receptor kinase (GRK) inhibitors. These results are the first to show, on endogenously expressed human delta-opioid receptor, that a close link exists between receptor phosphorylation and agonist-promoted desensitization and that desensitization involves a GRK.     

Molecular cloning and tissue expression of a novel orphan G protein-coupled receptor from rat lung

G protein-coupled receptors transduce the signal of a wide variety of hormones, neurotransmitters, cytokines, and other molecules across the cell membrane to elicit the corresponding response inside the target cells. We describe in this paper the molecular cloning and tissue distribution of a novel rat G protein-coupled receptor, GPR41, with highest homology to the human orphan G protein-coupled receptor DRY12. A lower degree of homology was seen with the receptors for bradykinin, angiotensin, and IL8. The expression of GPR41 appears to be the highest in brain and lung tissues, with lesser expression in heart, skeletal muscle, and kidney, as assayed by northern blotting. No GPR41 message was seen in spleen, liver, or testes. GPR41 failed to bind any of the ligands tested.     

Three-dimensional ultrastructural analysis of the Saccharomyces cerevisiae mitotic spindle

The three dimensional organization of microtubules in mitotic spindles of the yeast Saccharomyces cerevisiae has been determined by computer-aided reconstruction from electron micrographs of serially cross-sectioned spindles. Fifteen spindles ranging in length from 0.6-9.4 microns have been analyzed. Ordered microtubule packing is absent in spindles up to 0.8 micron, but the total number of microtubules is sufficient to allow one microtubule per kinetochore with a few additional microtubules that may form an interpolar spindle. An obvious bundle of about eight interpolar microtubules was found in spindles 1.3-1.6 microns long, and we suggest that the approximately 32 remaining microtubules act as kinetochore fibers. The relative lengths of the microtubules in these spindles suggest that they may be in an early stage of anaphase, even though these spindles are all situated in the mother cell, not in the isthmus between mother and bud. None of the reconstructed spindles exhibited the uniform populations of kinetochore microtubules characteristic of metaphase. Long spindles (2.7-9.4 microns), presumably in anaphase B, contained short remnants of a few presumed kinetochore microtubules clustered near the poles and a few long microtubules extending from each pole toward the spindle midplane, where they interdigitated with their counterparts from the other pole. Interpretation of these reconstructed spindles offers some insights into the mechanisms of mitosis in this yeast.     

G protein-coupled receptor kinase specificity for phosphorylation and desensitization of alpha2-adrenergic receptor subtypes

The alpha2-adrenergic receptor (alpha2AR) subtype alpha2C10 undergoes rapid agonist-promoted desensitization which is due to phosphorylation of the receptor. One kinase that has been shown to phosphorylate alpha2C10 in an agonist-dependent manner is the betaAR kinase (betaARK), a member of the family of G protein-coupled receptor kinases (GRKs). In contrast, the alpha2C4 subtype has not been observed to undergo agonist-promoted desensitization or phosphorylation by betaARK. However, the substrate specificities of the GRKs for phosphorylating alpha2AR subtypes are not known. We considered that differential capacities of various GRKs to phosphorylate alpha2C10 and alpha2C4 might be a key factor in dictating in a given cell the presence or extent of agonist-promoted desensitization of these receptors. COS-7 cells were co-transfected with alpha2C10 or alpha2C4 without or with the following GRKs: betaARK, betaARK2, GRK5, or GRK6. Intact cell phosphorylation studies were carried out by labeling cells with 32Pi, exposing some to agonist, and purifying the alpha2AR by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. BetaARK and betaARK2 were both found to phosphorylate alpha2C10 to equal extents (>2-fold over that of the endogenous kinases). On the other hand, GRK5 and GRK6 did not phosphorylate alpha2C10. In contrast to the findings with alpha2C10, alpha2C4 was not phosphorylated by any of these kinases. Functional studies carried out in transfected HEK293 cells expressing alpha2C10 or alpha2C4 and selected GRKs were consistent with these phosphorylation results. With the marked expression of these receptors, no agonist-promoted desensitization was observed in the absence of GRK co-expression. However, desensitization was imparted to alpha2C10 by co-expression of betaARK but not GRK6, while alpha2C4 failed to desensitize with co-expression of betaARK. These results indicate that short term agonist-promoted desensitization of alpha2ARs by phosphorylation is dependent on both the receptor subtype and the expressed GRK isoform.     

Isolation and chromosomal localization of GPR31, a human gene encoding a putative G protein-coupled receptor

The screening of a human genomic library with a chemokine receptor-like probe allowed us to obtain a putative member of the G protein-coupled receptor gene (GPCR) family, designated GPR31. Its deduced amino acid sequence encodes a polypeptide of 319 amino acids that shares 25-33% homology with members of the chemokine, purino, and somatostatin receptor gene families. Amino acid sequence comparison reveals that the best match in the protein databases is with the human orphan GPCR called HM74 (33% identity). Southern genomic analysis of the GPR31 gene shows a hybridization pattern consistent with that of a single-copy gene. Using fluorescence in situ hybridization, we have determined the chromosomal and regional localization of the GPR31 gene at 6q27. The GPR31 mRNA is expressed at low levels by several human cell lines of different cellular origins. The phylogenetic analysis suggests that the GPR31 receptor may represent a member of a new GPCR subfamily.     

Activation of the human estrogen receptor by estrogenic and antiestrogenic compounds in Saccharomyces cerevisiae: a positive selection system

OBJECTIVE: To develop a slow-release carboplatin formulation for intratumoral administration to cats. DESIGN: Preliminary study to analyze pharmacokinetic effects of purified sesame oil in the carboplatin formulation for intratumoral administration, and a second study to evaluate the efficacy and toxicosis of intratumoral administration of carboplatin in purified sesame oil. ANIMALS: 23 cats with squamous cell carcinomas of the nasal plane. PROCEDURE: Eight cats with advanced-stage tumors were submitted to intratumoral administration of 100 mg of carboplatin/m2 of body surface area, with or without purified sesame oil, using a two-period, cross-over design. Fifteen additional cats were treated by intratumoral administration of carboplatin in purified sesame oil. Four weekly intratumoral chemotherapy injections of carboplatin in purified sesame oil at a dosage of 1.5 mg/cm3 of tissue were given. RESULTS: Purified sesame oil in the formulation significantly reduced systemic exposure to carboplatin and drug leakage from the sites of injection. Cumulative effects of repeated intratumoral administrations on plasma concentrations of carboplatin were not observed. Systemic toxicosis was not observed, and local toxicosis was minimal. Healing of ulcerated lesions was not compromised. Rates of complete clinical tumor clearance and complete response were 67 and 73.3%, respectively. Product-limit estimates of mean progression-free survival times was 16 +/- 3.3 months. The 1-year progression-free survival rate was 55.1 +/- 13%. Local recurrence was observed in 7 cats; 4 had marginal tumor recurrence, and 3 had in-field and marginal tumor recurrence. CONCLUSIONS: Intratumoral carboplatin chemotherapy is safe and effective for cats with squamous cell carcinoma of the nasal plane. Future studies to improve treatment efficacy could include evaluation of increased dose-intensity as well as combination of this modality with radiotherapy. CLINICAL RELEVANCE: Intratumoral administration of carboplatin in a water-sesame-oil emulsion was found to be a practical and effective new treatment for facial squamous cell carcinomas in cats.     

Members of the G protein-coupled receptor kinase family that phosphorylate the beta2-adrenergic receptor facilitate sequestration

We recently reported that a beta2-adrenergic receptor (beta2AR) mutant, Y326A, defective in its ability to sequester in response to agonist stimulation was a poor substrate for G protein-coupled receptor kinase (GRK)-mediated phosphorylation; however, its ability to be phosphorylated and sequestered could be restored by overexpressing GRK2 [Ferguson et al. (1995) J. Biol. Chem. 270, 24782]. In the present report, we tested the ability of each of the known GRKs (GRK1-6) to phosphorylate and rescue the sequestration of the Y326A mutant in HEK-293 cells. We demonstrate that in addition to GRK2, GRK3-6 can phosphorylate the Y326A mutant and rescue its sequestration; however, GRK1 was totally ineffective in rescuing either the phosphorylation or the sequestration of the mutant receptor. We found that the agonist-dependent rescue of Y326A mutant phosphorylation by GRK2, -3, and -5 was associated with the agonist-dependent rescue of sequestration. In contrast, overexpression of GRK4 and -6 led mainly to agonist-independent phosphorylation of the Y326A mutant accompanied by increased basal receptor sequestration. Our results demonstrate that phosphorylation per se, but not the interaction with a specific GRK, is required to facilitate beta2AR sequestration.     

Composition and functional analysis of the Saccharomyces cerevisiae trehalose synthase complex

In the yeast Saccharomyces cerevisiae, trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP), which convert glucose 6-phosphate plus UDP-glucose to trehalose, are part of the trehalose synthase complex. In addition to the TPS1 (previously also called GGS1, CIF1, BYP1, FDP1, GLC6, and TSS1) and TPS2 (also described as HOG2 and PFK3) gene products, this complex also contains a regulatory subunit encoded by TSL1. We have constructed a set of isogenic strains carrying all possible combinations of deletions of these three genes and of TPS3, a homologue of TSL1 identified by systematic sequencing. Deletion of TPS1 totally abolished TPS activity and measurable trehalose, whereas deletion of any of the other genes in most cases reduced both. Similarly, deletion of TPS2 completely abolished TPP activity, and deletion of any of the other genes resulted in a reduction of this activity. Therefore, it appears that all subunits are required for optimal enzymatic activity. Since we observed measurable trehalose in strains lacking all but the TPS1 gene, some phosphatase activity in addition to Tps2 can hydrolyze trehalose 6-phosphate. Deletion of TPS3, in particular in a tsl1Delta background, reduced both TPS and TPP activities and trehalose content. Deletion of TPS2, TSL1, or TPS3 and, in particular, of TSL1 plus TPS3 destabilized the trehalose synthase complex. We conclude that Tps3 is a fourth subunit of the complex with functions partially redundant to those of Tsl1. Among the four genes studied, TPS1 is necessary and sufficient for growth on glucose and fructose. Even when overproduced, none of the other subunits could take over this function of Tps1 despite the homology shared by all four proteins. A portion of Tps1 appears to occur in a form not bound by the complex. Whereas TPS activity in the complex is inhibited by Pi, Pi stimulates the monomeric form of Tps1. We discuss the possible role of differentially regulated Tps1 in a complex-bound or monomeric form in light of the requirement of Tps1 for trehalose production and for growth on glucose and fructose.     

Palmitoylation increases the kinase activity of the G protein-coupled receptor kinase, GRK6

The G protein-coupled receptor kinase GRK6 undergoes posttranslational modification by palmitoylation. Palmitoylated GRK6 is associated with the membrane, while nonpalmitoylated GRK6 remains cytosolic. We have separated palmitoylated from nonpalmitoylated GRK6 to assess their relative kinase activity. Palmitoylated GRK6 is 10-fold more active at phosphorylating beta2-adrenergic receptor than nonpalmitoylated wild-type GRK6 or a nonpalmitoylatable mutant GRK6. A nonpalmitoylatable mutant GRK6 which has been further mutated to undergo posttranslational geranylgeranylation is also more active, recovering most of the activity of the palmitoylated enzyme. This activity increase by lipid modification is expected, as the lipid helps GRK6 localize to cellular membranes where its receptor substrates are found. However, when assayed using a soluble protein (casein) as a substrate, both palmitoylated and prenylated GRK6 display significantly higher activity than nonpalmitoylated wild-type or nonpalmitoylatable mutant GRK6 kinases. This increased activity is not altered by addition of exogenous palmitate or phosphatidycholine vesicles, arguing that it is not due to direct activation of GRK6 by binding palmitate, nor to nonspecific association of the GRK6 with casein. Further, chemical depalmitoylation reduces the casein phosphorylation activity of the palmitoylated, but not prenylated, GRK6 kinase. Thus, palmitoylation of GRK6 appears to play a dual role in increasing the activity of GRK6: it increases the hydrophobicity and membrane association of the GRK6 protein, which helps bring the GRK6 to its membrane-bound substrates, and it increases the kinase catalytic activity of GRK6.     

Molecular cloning and expression of an avian G protein-coupled P2Y receptor

A family of G protein-coupled P2Y receptors that are activated by adenine and uridine nucleotides has been identified recently. Degenerate primers based on conserved sequences in these P2Y receptors were used to amplify turkey DNA, which was used to isolate the complete coding sequence of a cDNA that encodes a novel G protein-coupled receptor. Stable expression of this avian cDNA in 1321N1 human astrocytoma cells resulted in the conveyance of marked inositol phosphate responses to various nucleotides. Although this cloned avian receptor exhibited its highest homology to the previously cloned mammalian P2Y4 receptor, its pharmacological selectivity was not consistent with the avian receptor's being a species homologue of the P2Y4 receptor. That is, whereas the P2Y4 receptor is selectively activated by UTP and is not activated by ATP or Ap4A, the novel avian receptor was potently activated by ATP and Ap4A as well as by UTP. Taken together, these results describe the identification of an avian phospholipase C-coupled P2Y receptor that, like the mammalian P2Y2 receptor, is activated by both adenine and uridine nucleotides.