Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

FSH rapidly desensitizes the FSH-receptor (FSH-R) upon binding. Very little information is available concerning the regulatory proteins involved in this process. In the present study, we investigated whether G protein-coupled receptor kinases (GRKs) and arrestins have a role in FSH-R desensitization, using a mouse Ltk 7/12 cell line stably overexpressing the rat FSH-R as a model. We found that these cells, which express GRK2, GRK3, GRK5, and GRK6 as well as beta-arrestins 1 and 2 as detected by RT-PCR and by Western blotting, were rapidly desensitized in the presence of FSH. Overexpression of GRKs and/or beta-arrestins in Ltk 7/12 cells allowed us to demonstrate 1) that GRK2, -3, -5, -6a, and -6b inhibit the FSH-R-mediated signaling (from 71% to 96% of maximal inhibition depending on the kinase, P < 0.001); 2) that beta-arrestins 1 or 2 also decrease the FSH action when overexpressed (80% of maximal inhibition, P < 0.01) whereas dominant negative beta-arrestin 2 [319-418] potentiates it 8-fold (P < 0.001); 3) that beta-arrestins and GRKs (except GRK6a) exert additive inhibition on FSH-induced response; and 4) that FSH-R desensitization depends upon the endogenous expression of GRKs, since there is potentiation of the FSH response (2- to 3-fold, P < 0.05) with antisenses cDNAs for GRK2, -5, and -6, but not GRK3. Our results show that the desensitization of the FSH-induced response involves the GRK/arrestin system.
Mol Endocrinol 1999 Sep
PMID:Involvement of G protein-coupled receptor kinases and arrestins in desensitization to follicle-stimulating hormone action. 1047 49

The alpha-mating pheromone receptor encoded by the STE2 gene of the yeast Saccharomyces cerevisiae is a G protein-coupled receptor (GPCR) that is homologous to the large family of GPCRs that mediate multiple types of signal transduction in mammals. We have screened libraries of mutant receptors to identify dominant negative alleles that are capable of interfering with the function of a co-expressed normal receptor. Two dominant negative alleles have been recovered in this manner. In addition, we find that previously isolated loss-of-function mutations in the alpha-factor receptor exhibit dominant negative effects. Detection of the dominant effects requires high-level expression of the mutant receptors but does not require a high ratio of mutant to normal receptors. Cellular levels of the normal receptors are not affected by co-expression of the dominant negative alleles. Expression of the mutant receptors does not interfere with constitutive signaling in a strain that lacks the G protein alpha subunit encoded by GPA1, indicating that interference with signaling occurs at the level of the receptor or the interacting G protein. Expression of increased levels of G protein subunits partially reverses the dominant negative effects. The dominant negative behavior of the mutant receptors is diminished by deletion of the SST2 gene, which encodes an RGS (Regulator of G protein Signaling) protein involved in desensitization of pheromone signaling. The most likely explanation for the dominant negative effects of the mutations appears to be the existence of an interaction between unactivated receptors and the trimeric G protein that titrates the G protein away from the normal receptors or renders the G protein insensitive to receptor activation. This interaction appears to be mediated by the SST2 gene product.
Mol Gen Genet 1999 Jul
PMID:Dominant negative mutations in the alpha-factor receptor, a G protein-coupled receptor encoded by the STE2 gene of the yeast Saccharomyces cerevisiae. 1048 82

Fusion proteins were generated between the human 5-hydroxytryptamine (5-HT)(1A) receptor and both wild-type (Cys(351)) and pertussis toxin-resistant (Gly(351) and Ile(351)) forms of G(i1). These were expressed stably. Pertussis toxin treatment substantially reduced basal high-affinity GTPase activity in clones expressing the 5-HT(1A) receptor wild-type G(i1)alpha construct but not in clones expressing 5-HT(1A) receptor (Gly(351))G(i1)alpha or (Ile(351))G(i1)alpha. Spiperone functioned as an inverse agonist in membranes expressing the 5-HT(1A) receptor wild-type G(i1)alpha fusion protein and in those expressing 5-HT(1A) receptor (Ile(351))G(i1)alpha but not the 5-HT(1A) receptor (Gly(351))G(i1)alpha fusion protein. The effect of spiperone at the 5-HT(1A) receptor wild-type G(i1)alpha construct but not the 5-HT(1A) receptor (Ile(351))G(i1)alpha construct was blocked by pertussis toxin treatment. By contrast, agonists functioned with equal effectiveness at the three fusion proteins and were unaffected by pertussis toxin treatment of the (Ile(351))G(i1)alpha- and (Gly(351))G(i1)alpha-containing constructs. 5-HT resulted in strong inhibition of forskolin-amplified adenylyl cyclase in intact cells expressing the isolated 5-HT(1A) receptor. In fusion protein-expressing cells, 5-HT-mediated inhibition of adenylyl cyclase was also observed. Pertussis toxin treatment obliterated 5-HT-mediated inhibition in cells expressing the isolated receptor and the 5-HT(1A) receptor wild-type G(i1)alpha fusion protein but not in those expressing the 5-HT(1A) receptor (Ile(351)) or (Gly(351))G(i1)alpha fusion proteins. These studies demonstrate that alteration of a single amino acid in G(i1)alpha located at a key contact site between the G protein and a G protein-coupled receptor can regulate agonist-independent constitutive activity of the G protein-coupled receptor and that fusion proteins can directly regulate adenylyl cyclase.
Mol Pharmacol 1999 Oct
PMID:Regulation of G protein activation and effector modulation by fusion proteins between the human 5-hydroxytryptamine(1A) receptor and the alpha subunit of G(i1): differences in receptor-constitutive activity imparted by single amino acid substitutions in G(i1)alpha. 1049 50

Studies of the desensitization of G protein-coupled signal transduction have led to the discovery of a family of guanosine triphosphatase-activating proteins (GAPs) for heterotrimeric G protein alpha subunits - the "regulator of G protein signaling" or RGS proteins. In considering both documented and potential functions of several RGS protein family members with demonstrable multidomain compositions (p115RhoGEF, PDZRhoGEF, Axin, Axil/Conductin, D-AKAP2, the G protein-coupled receptor kinases [GRKs], the DEP/GGL/RGS subfamily [RGS6, RGS7, RGS9, RGS11], and RGS12), this review explores the shift in our appreciation of the RGS proteins from unidimensional desensitizing agents to multifocal signal transduction regulators.
Crit Rev Biochem Mol Biol 1999
PMID:Whither goest the RGS proteins? 1051 44

The mu opioid receptor, a primary site of action in the brain for opioid neuropeptides and opiate drugs of abuse, is a member of the seven transmembrane, G protein-coupled receptor (GPCR) superfamily. Two cysteine residues, one in each of the first two of three extracellular loops (ECLs), are highly conserved among GPCRs, and there is direct or circumstantial evidence that the residues form a disulfide bond in many of these receptors. Such a bond would dramatically govern the topology of the ECLs, and possibly affect the position of the membrane-spanning domains. Recent findings from several laboratories indicate the importance of the ECLs for opioid ligand selectivity. These conserved cysteine residues in the mu opioid receptor were studied using site-directed mutagenesis. Little or no specific binding of radiolabled opiate alkaloid or opioid peptide agonists or antagonists was observed for receptors mutated at either "disulfide cysteine" residue. Each mutant mu opioid receptor was expressed in both transiently- and stably-transfected cells, in some cases at levels comparable to the wild type receptor. The two point mutants possessing serine-for-cysteine substitutions were also observed to successfully reach the cell plasma membrane, as evidenced by electron microscopy. Consistent with related work with other GPCRs, the mu opioid receptor apparently also employs the extracellular disulfide bond. This information now permits accurate molecular modeling of extracellular aspects of the receptor, including plausible scenarios of mu receptor docking of opioid ligands known to require specific extracellular loop features for high affinity binding.
Brain Res Mol Brain Res 1999 Oct 01
PMID:Mutation of human mu opioid receptor extracellular "disulfide cysteine" residues alters ligand binding but does not prevent receptor targeting to the cell plasma membrane. 1052 78

Synthetic ligands have been identified that reset and amplify the cycle of pulsatile GH secretion by interacting with the orphan GH-secretagogue receptor (GHS-R). The GHS-R is rhodopsin like, but does not obviously belong to any of the established G protein-coupled receptor (GPCR) subfamilies. We recently characterized the closely related orphan family member, GPR38, as the motilin receptor. A common property of both receptors is that they amplify and sustain pulsatile biological responses in the continued presence of their respective ligands. To efficiently identify additional members of this new GPCR family, we explored a vertebrate species having a compact genome, that was evolutionary distant from human, but where functionally important genes were likely to be conserved. Accordingly, three distinct full-length clones, encoding proteins of significant identity to the human GHS-R, were isolated from the Pufferfish (Spheroides nephelus). Southern analyses showed that the three cloned Pufferfish genes are highly conserved across species. The gene with closest identity (58%) was activated by three synthetic ligands that were chosen for their very high selectivity on the GHS-R as illustrated by their specificity in activating the wild-type human GHS-R but not the E124Q mutant. These results indicate that the ligand activation domain of the GHS-R has been evolutionary conserved from Pufferfish to human (400 million years), supporting the notion that the GHS-R and its natural ligand play a fundamentally important role in biology. Furthermore, they illustrate the power of exploiting the compact Pufferfish genome for simplifying the isolation of endocrinologically important receptor families.
Mol Endocrinol 2000 Jan
PMID:Ligand activation domain of human orphan growth hormone (GH) secretagogue receptor (GHS-R) conserved from Pufferfish to humans. 1062 55

Hundreds of extracellular stimuli are received by cells via the pathways consisting of three basic components: cell-surface receptors, heterotrimeric G proteins, and intracellular effector enzymes or ion channels. A number of additional molecules, including G protein-coupled receptor kinases (GRKs), phosducin and Ca(2+)-binding proteins modulate signal transduction through these cascades. Understanding how these universal pathways work requires a detailed analysis of the interactions between these proteins. The recently emerged technology of surface plasmon resonance (SPR) can study protein-protein interactions by measuring not only the equilibrium binding constants, but also the association and dissociation rates. This article reviews experimental design used by researchers to analyze different components of the G protein pathway by SPR and focuses on the insights this technique provides regarding the kinetics, structure-function aspects and regulation of specific molecular events in the cascade.
J Mol Recognit
PMID:Application of surface plasmon resonance for analysis of protein-protein interactions in the G protein-mediated signal transduction pathway. 1067 93

A site-directed mutagenesis approach has been used to gain insight into the molecular events whereby the heptadecapeptide nociceptin binds and activates the opioid receptor-like 1 (ORL1) receptor, a G protein-coupled receptor. Alanine mutation, in the human ORL1 receptor, of transmembrane amino acid residues that are conserved in opioid receptors, Asp(130) and Tyr(131) in transmembrane segment (TM) III, Phe(220) and Phe(224) in TM V, and Trp(276) in TM VI, yields mutant receptors with reduced affinity, and proportionally decreased reactivity, toward nociceptin. Least to most deleterious in this respect are Ala substitutions of Phe(220) approximately W276A < Tyr(131) << Phe(224) </= Asp(130). The dramatic effects of the D130A mutation on nociceptin binding and activity are not reversed in the D130N mutant, whereas those of the Y131A mutation are totally suppressed in Y131F. This suggests that a negative charge at position 130, and a phenyl ring at position 131 in TM III, are critical for occupancy and/or activation of the receptor by nociceptin. Alanine replacement of glutamine 286, located at the C terminus of TM VI, yields a mutant receptor that binds nociceptin with nearly the same affinity as does the wild-type receptor (K(d) values of 0.13 and 0.22 nM, respectively) but, unlike the latter, is unable to mediate nociceptin inhibition of forskolin-induced cAMP synthesis in recombinant Chinese hamster ovary cells (ED(50) > 10,000 nM compared with 0.8 nM at the wild-type receptor). In all respects, this mutant receptor appears to be functionally inactive, indicating that residue Gln(286) may play a pivotal role in ORL1 receptor-mediated transduction of the nociceptin signal.
Mol Pharmacol 2000 Mar
PMID:Functional inactivation of the nociceptin receptor by alanine substitution of glutamine 286 at the C terminus of transmembrane segment VI: evidence from a site-directed mutagenesis study of the ORL1 receptor transmembrane-binding domain. 1069 89

The regulation of the cellular distribution and intracellular signaling properties of the alpha(1B)- and alpha(1D)- adrenoceptor (alpha(1)-AR) subtypes was examined in stably transfected Rat 1 fibroblasts. In unstimulated cells, alpha(1B)-AR expression was noted primarily on the cell surface. Treatment with phenylephrine induced internalization of the alpha(1B)-AR and promoted association with arrestin 2. The internalized alpha(1B)-AR colocalized with the transferrin receptor, an endosomal marker. In unstimulated fibroblasts, the alpha(1D)-AR was detected in a perinuclear orientation and was colocalized with arrestin 2 in a compartment also containing the transferrin receptor. After treatment with prazosin, which exhibits inverse agonist properties, the alpha(1D)-AR was redistributed from intracellular sites to the cellular periphery and was no longer associated with the transferrin receptor or arrestin 2. alpha(1D)-AR-expressing cells exhibited a high degree of basal activity for both inositol phosphate formation and extracellular signal regulated kinase (ERK), which was reduced by treatment with prazosin. In these cells, phenylephrine induced a dose-dependent increase in inositol phosphate formation but had no effect on ERK activity. In alpha(1B) -AR-expressing cells, phenylephrine stimulated both inositol phosphate formation and ERK activity. These data show that: 1) there are differences in the cellular localization of the alpha(1)-AR subtypes; 2) the alpha(1B)-AR exhibits expected G protein-coupled receptor activity regarding cellular localization, agonist-mediated internalization, and coupling to second messengers; and 3) the alpha(1D)-AR is constitutively active and, as a result, is localized to intracellular compartments involved in receptor recycling.
Mol Pharmacol 2000 Apr
PMID:Regulation of the cellular localization and signaling properties of the alpha(1B)- and alpha(1D)-adrenoceptors by agonists and inverse agonists. 1072 10

A variety of G protein-coupled receptors (GPCRs) are phosphorylated by G protein-coupled receptor kinase 2 (GRK2). This event promotes the binding of regulatory proteins termed beta-arrestins to GPCRs, leading to uncoupling from G proteins and receptor internalization. Recent data indicate that GRK2 and beta-arrestins also play an important role in the stimulation of the extracellular signal-regulated kinases (ERK)/mitogen-activated protein kinase (MAPK) cascade by GPCRs. In this report, we have investigated the existence of functional interactions between GRK2 and MAPK. We show that activation of beta(2)-adrenergic receptors (beta(2)-AR) promotes the rapid association of GRK2 and MAPK in living cells, as assessed by coimmunoprecipitation experiments in COS-7 cells transfected with beta(2)-AR, GRK2, and an epitope-tagged MAPK. Coimmunoprecipitation of MAPK and GRK2 is blocked by inhibition of the MAPK cascade and is not observed upon activation of MAPK in the absence of beta(2)-AR stimulation, thus indicating that both an active MAPK and agonist occupancy of GPCR are required for the association to occur. Interestingly, we have found that purified ERK1/MAPK can directly phosphorylate the C-terminal domain of GRK2, and that the phosphorylation process is favored by the presence of Gbetagamma-subunits or an activated receptor. Furthermore, GRK2 phosphorylation by MAPK leads to a decreased activity of GRK2 toward GPCR. Taken together, our results suggest that stimulation of GPCRs promotes the rapid association of GRK2 and MAPK leading to modulation of GRK2 functionality, thus putting forward a new feedback mechanism for the regulation of GPCR signaling.
Mol Pharmacol 2000 Apr
PMID:Agonist-dependent modulation of G protein-coupled receptor kinase 2 by mitogen-activated protein kinases. 1072 25


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