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1.
The selectivity in coupling of various receptors to GTP-binding regulatory proteins (G proteins) was examined directly by a novel assay entailing the use of proteins overexpressed in Spodoptera frugiperda (Sf9) cells. Activation of G proteins was monitored in membranes prepared from Sf9 cells co-expressing selected pairs of receptors and G proteins (i.e. alpha, beta1, and gamma2 subunits). Membranes were incubated with [35S]guanosine 5'-(3-O-thio)triphosphate (GTPgammaS) +/- an agonist, and the amount of radiolabel bound to the alpha subunit was quantitated following immunoprecipitation. When expressed without receptor (but with beta1gamma2), the G protein subunits alphaz, alpha12, and alpha13 did not bind appreciable levels of [35S]GTPgammaS, consistent with a minimal level of GDP/[35S]GTPgammaS exchange. In contrast, the subunits alphas and alphaq bound measurable levels of the nucleotide. Co-expression of the 5-hydroxytryptamine1A (5-HT1A) receptor promoted binding of [35S]GTPgammaS to alphaz but not to alpha12, alpha13, or alphas. Binding to alphaz was enhanced by inclusion of serotonin in the assay. Agonist activation of both thrombin and neurokinin-1 receptors promoted a modest increase in [35S]GTPgammaS binding to alphaz and more robust increases in binding to alphaq, alpha12, and alpha13. Binding of [35S]GTPgammaS to alphas was strongly enhanced only by the activated beta1-adrenergic receptor. Our data identify interactions of receptors and G proteins directly, without resort to measurements of effector activity, confirm the coupling of the 5-HT1A receptor to Gz and extend the list of receptors that interact with this unique G protein to the receptors for thrombin and substance P, imply constitutive activity for the 5-HT1A receptor, and demonstrate for the first time that the cloned receptors for thrombin and substance P activate G12 and G13.  相似文献   

2.
Signaling pathways that link extracellular factors to activation of the monomeric guanosine triphosphatase (GTPase) Rho control cytoskeletal rearrangements and cell growth. Heterotrimeric guanine nucleotide-binding proteins (G proteins) participate in several of these pathways, although their mechanisms are unclear. The GTPase activities of two G protein alpha subunits, Galpha12 and Galpha13, are stimulated by the Rho guanine nucleotide exchange factor p115 RhoGEF. Activated Galpha13 bound tightly to p115 RhoGEF and stimulated its capacity to catalyze nucleotide exchange on Rho. In contrast, activated Galpha12 inhibited stimulation by Galpha13. Thus, p115 RhoGEF can directly link heterotrimeric G protein alpha subunits to regulation of Rho.  相似文献   

3.
Pertussis toxin inhibits chemotaxis of neutrophils by preventing chemoattractant receptors from activating trimeric G proteins in the Gi subfamily. In HEK293 cells expressing recombinant receptors, directional migration toward appropriate agonist ligands requires release of free G protein betagamma subunits and can be triggered by agonists for receptors coupled to Gi but not by agonists for receptors coupled to two other G proteins, Gs and Gq. Because activation of any G protein presumably releases free Gbetagamma, we tested the hypothesis that chemotaxis also requires activated alpha subunits (Galphai) of Gi proteins. HEK293 cells were stably cotransfected with the Gi-coupled receptor for interleukin-8, CXCR1, and with a chimeric Galpha, Galphaqz5, which resembles Galphai in susceptibility to activation by Gi-coupled receptors but cannot regulate the Galphai effector, adenylyl cyclase. These cells, unlike cells expressing CXCR1 alone, migrated toward interleukin-8 even after treatment with pertussis toxin, which prevents activation of endogenous Galphai but not that of Galphaqz5. We infer that chemotaxis does not require activation of Galphai. Because chemotaxis is mediated by Gbetagamma subunits released when Gi-coupled receptors activate Galphaqz5, but not when Gq- or Gs-coupled receptors activate their respective G proteins, we propose that Gi-coupled receptors transmit a necessary chemotactic signal that is independent of Galphai.  相似文献   

4.
Cell surface receptors that mediate endocytosis cluster into clathrin-coated pits, which pinch off to form vesicles that transport the receptors and their ligands. This multi-step process requires the coordinated action of many factors, including GTP-hydrolyzing proteins such as dynamin and regulators of actin cytoskeleton assembly. We note herein that sequestration of heterotrimeric G protein beta gamma subunits in intact cells strongly inhibits clathrin-coated pit-mediated endocytosis and causes rearrangement of the actin cytoskeleton. Our results suggest that cells contain a pool of free beta gamma and that it functions constitutively to permit endocytosis.  相似文献   

5.
RGS10 is a selective activator of G alpha i GTPase activity   总被引:1,自引:0,他引:1  
Polypeptides that define a protein family termed RGS (for regulators of G-protein signalling) are encoded by the SST2 gene of the yeast Saccharomyces cerevisiae, the EGL-10 gene of the nematode Caenorhabdatis elegans, and several related mammalian genes. Genetic studies in invertebrates and mammalian cell-transfection experiments indicate that RGS proteins negatively regulate signalling pathways involving seven transmembrane receptors and heterotrimeric G proteins. However, the biochemical mechanism by which RGS proteins control these pathways is unknown. Here we report the characterization of human RGS10, a member of this protein family. Co-immunoprecipitation studies demonstrate that RGS10 associates specifically with the activated forms of two related G-protein subunits, G alphai3, and G alphaz, but fails to interact with the structurally and functionally distinct G alphas subunit. In vitro assays with purified proteins indicate that RGS10 increases potently and selectively the GTP hydrolytic activity of several members of the G alphai family, including G alphai3, G alphaz, and G alpha0. These results demonstrate that RGS proteins can attenuate signalling pathways involving heterotrimeric G proteins by serving as GTPase-activating proteins for specific types of G alpha subunits.  相似文献   

6.
We have compared the sedimentation rates on sucrose gradients of the heterotrimeric GTP-binding regulatory (G) proteins Gs, G(o), Gi, and Gq extracted from rat brain synaptoneurosomes with Lubrol and digitonin. The individual alpha and beta subunits were monitored with specific antisera. In all cases, both subunits cosedimented, indicating that the subunits are likely complexed as heterotrimers. When extracted with Lubrol all of the G proteins sedimented with rates of about 4.5 S (consistent with heterotrimers) whereas digitonin extracted 60% of the G proteins with peaks at 11 S; 40% pelleted as larger structures. Digitonin-extracted Gi was cross-linked by p-phenylenedimaleimide, yielding structures too large to enter polyacrylamide gels. No cross-linking of Lubrol-extracted Gi occurred. Treatment of the membranes with guanosine 5'-[gamma-thio]triphosphate and Mg2+ yielded digitonin-extracted structures with peak sedimentation values of 8.5 S--i.e., comparable to that of purified G(o) in digitonin and considerably larger than the Lubrol-extracted 2S structures representing the separated alpha and beta gamma subunits formed by the actions of guanosine 5'-[gamma-thio]triphosphate. It is concluded that the multimeric structures of G proteins in brain membranes are at least partially preserved in digitonin and that activation of these structures in membranes yields monomers of G proteins rather than the disaggregated products (alpha and beta gamma complexes) observed in Lubrol. It is proposed that hormones and GTP affect the dynamic interplay between multimeric G proteins and receptors in a fashion analogous to the actions of ATP on the dynamic interactions between myosin and actin filaments. Signal transduction is mediated by activated monomers released from the multimers during the activation process.  相似文献   

7.
p115 RhoGEF, a GTPase activating protein for Galpha12 and Galpha13   总被引:1,自引:0,他引:1  
Members of the regulators of G protein signaling (RGS) family stimulate the intrinsic guanosine triphosphatase (GTPase) activity of the alpha subunits of certain heterotrimeric guanine nucleotide-binding proteins (G proteins). The guanine nucleotide exchange factor (GEF) for Rho, p115 RhoGEF, has an amino-terminal region with similarity to RGS proteins. Recombinant p115 RhoGEF and a fusion protein containing the amino terminus of p115 had specific activity as GTPase activating proteins toward the alpha subunits of the G proteins G12 and G13, but not toward members of the Gs, Gi, or Gq subfamilies of Galpha proteins. This GEF may act as an intermediary in the regulation of Rho proteins by G13 and G12.  相似文献   

8.
Recombinant regulators of G protein-signaling (RGS) proteins stimulate hydrolysis of GTP by alpha subunits of the Gi family but have not been reported to regulate other G protein alpha subunits. Expression of recombinant RGS proteins in cultured cells inhibits Gi-mediated hormonal signals probably by acting as GTPase-activating proteins for Galphai subunits. To ask whether an RGS protein can also regulate cellular responses mediated by G proteins in the Gq/11 family, we compared activation of mitogen-activated protein kinase (MAPK) by a Gq/11-coupled receptor, the bombesin receptor (BR), and a Gi-coupled receptor, the D2 dopamine receptor, transiently co-expressed with or without recombinant RGS4 in COS-7 cells. Pertussis toxin, which uncouples Gi from receptors, blocked MAPK activation by the D2 dopamine receptor but not by the BR. Co-expression of RGS4, however, inhibited activation of MAPK by both receptors causing a rightward shift of the concentration-effect curve for both receptor agonists. RGS4 also inhibited BR-stimulated synthesis of inositol phosphates by an effector target of Gq/11, phospholipase C. Moreover, RGS4 inhibited inositol phosphate synthesis activated by addition of AlF4- to cells overexpressing recombinant alphaq, probably by binding to alphaq.GDP.AlF4-. These results demonstrate that RGS4 can regulate Gq/11-mediated cellular signals by competing for effector binding as well as by acting as a GTPase-activating protein.  相似文献   

9.
Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) serve to transduce information from agonist-bound receptors to effector enzymes or ion channels. Current models of G protein activation-deactivation indicate that the oligomeric GDP-bound form must undergo release of GDP, bind GTP and undergo subunit dissociation, in order to be in active form (GTP bound alpha subunits and free beta gamma dimers) and to regulate effectors. The effect of receptor occupation by an agonist is generally accepted to be promotion of guanine nucleotide exchange thus allowing activation of the G protein. Recent studies indicate that transphosphorylation leading to the formation of GTP from GDP and ATP in the close vicinity, or even at the G protein, catalysed by membrane-associated nucleoside diphosphate kinase, may further activate G proteins. This activation is demonstrated by a decreased affinity of G protein-coupled receptors for agonists and an increased response of G protein coupled effectors. In addition, a phosphorylation of G protein beta subunits and consequent phosphate transfer reaction resulting in G protein activation has also been demonstrated. Finally, endogenously formed GTP was preferentially effective in activating some G proteins compared to exogenous GTP. The aim of this report is to present an overview of the evidence to date for a transphosphorylation as a means of G protein activation (see also refs [1 and 2] for reviews).  相似文献   

10.
Several G protein-coupled receptors that interact with pertussis toxin-sensitive heterotrimeric G proteins mediate Ras-dependent activation of mitogen-activated protein (MAP) kinases. The mechanism involves Gbetagamma subunit-mediated increases in tyrosine phosphorylation of the Shc adapter protein, Shc*Grb2 complex formation, and recruitment of Ras guanine nucleotide exchange factor activity. We have investigated the role of the ubiquitous nonreceptor tyrosine kinase c-Src in activation of the MAP kinase pathway via endogenous G protein-coupled lysophosphatidic acid (LPA) receptors or by transient expression of Gbetagamma subunits in COS-7 cells. In vitro kinase assays of Shc immunoprecipitates following LPA stimulation demonstrated rapid, transient recruitment of tyrosine kinase activity into Shc immune complexes. Recruitment of tyrosine kinase activity was pertussis toxin-sensitive and mimicked by cellular expression of Gbetagamma subunits. Immunoblots for coprecipitated proteins in Shc immunoprecipitates revealed a transient association of Shc and c-Src following LPA stimulation, which coincided with increases in Shc-associated tyrosine kinase activity and Shc tyrosine phosphorylation. LPA stimulation or expression of Gbetagamma subunits resulted in c-Src activation, as assessed by increased c-Src autophosphorylation. Overexpression of wild-type or constitutively active mutant c-Src, but not kinase inactive mutant c-Src, lead to increased tyrosine kinase activity in Shc immunoprecipitates, increased Shc tyrosine phosphorylation, and Shc.Grb2 complex formation. MAP kinase activation resulting from LPA receptor stimulation, expression of Gbetagamma subunits, or expression of c-Src was sensitive to dominant negatives of mSos, Ras, and Raf. Coexpression of Csk, which inactivates Src family kinases by phosphorylating the regulatory C-terminal tyrosine residue, inhibited LPA stimulation of Shc tyrosine phosphorylation, Shc.Grb2 complex formation, and MAP kinase activation. These data suggest that Gbetagamma subunit-mediated formation of Shc.c-Src complexes and c-Src kinase activation are early events in Ras-dependent activation of MAP kinase via pertussis toxin-sensitive G protein-coupled receptors.  相似文献   

11.
Among membrane-bound receptors, the seven transmembrane receptors are the most abundant (several thousand, 1% of the genome). They were the most successful during evolution. They are capable of transducing messages as different as photons, organic odorants, nucleotides, nucleosides, peptides, lipids, proteins, etc. They are catalysts of the GDP/GTP nucleotide exchange on heterotrimeric G proteins. They are therefore also called 'G-protein-coupled receptors' (GPCR). G proteins are composed of three subunits, G alpha and two undissociable subunits, G beta gamma. There are at least three families of GPCR showing no sequence similarity. Among G proteins, some have been crystallized (including under the heterotrimeric form) and their structure as well as their activation mechanisms are well known. The structures of GPCR are less known owing to the difficulty in crystallizing membrane-bound proteins. Indirect studies (mutations, 2D crystallization of rhodopsine, molecular modelling, etc.) lead to a useful model of the 'central core' composed of the seven transmembrane domains and of its structural modifications during activation. The intimate contact zones between GPCR and G proteins include, on the GPCR side, domains of intracellular loops and C-terminal, which are specific for each family and on the G protein side, essentially the N- et C-terminal domains plus the alpha 4-beta 6 loop. GPCR can adopt several 'active' conformations some of them being found in mutated receptors responsible for pathologies.  相似文献   

12.
Members of the beta isozyme subfamily of phosphatidylinositol-specific phospholipase C (PLC) are stimulated by alpha subunits and betagamma dimers of heterotrimeric guanine-nucleotide-binding proteins (G proteins). Myeloid differentiated human HL-60 granulocytes and bovine neutrophils contain a soluble phospholipase C, which is stimulated by the metabolically stable GTP analogue guanosine (5'-->O)-3-thiotriphosphate (GTP[S]). To identify the component(s) involved in mediating this stimulation, the relevant polypeptide(s) was resolved from endogenous phospholipase C and purified from bovine neutrophil cytosol by measuring its ability to confer GTP[S] stimulation to exogenous recombinant PLCbeta2. The resolved factor, which behaved as 48-kDa protein upon gel filtration, stimulated PLCbeta2 but not PLCbeta1 or PLCdelta1. Activation of phosphatidylinositol 4-phosphate 5-kinase was not involved in this stimulation. The purified stimulatory factor consisted of two polypeptides of molecular masses of approximately 23 kDa and 26 kDa. The protein stimulated a deletion mutant of PLCbeta2 that lacked a carboxyl-terminal region necessary for stimulation by members of the alpha(q) subfamily of the G-protein alpha subunits. The results of this study suggest that a GTP-binding protein distinct from alpha(q) subunits, probably a low-molecular-mass GTP-binding protein associated with a regulatory protein, is involved in isozyme-specific activation of PLCbeta2.  相似文献   

13.
The heterotrimeric G proteins are often regarded functionally as a heterodimer, consisting of a guanine nucleotide-binding alpha subunit and a beta gamma subunit complex. Since the tightly associated beta gamma subunit complex can be separated only under denaturing conditions, studies aimed at determining the individual contributions of the beta and gamma subunits in terms of binding to the various alpha subunits, interacting with receptors, and regulating effectors, have not been possible. To circumvent this problem, we have used baculovirus-infected cells to direct the individual expression of the beta 1 and gamma 2 subunits. Application of extracts from baculovirus-infected cells to an alpha subunit of G protein (G(o) alpha)-affinity matrix resulted in the selective retention and AMF-specific elution of the expressed gamma 2 subunit, but not the expressed beta 1 subunit. Overall, these and other data provide the first evidence of a direct association between the gamma and alpha subunits, which is dependent on prenylation of gamma. The apparent direct association between the gamma and alpha subunits was further probed by limited trypsin proteolysis. Upon addition of trypsin, the G(o) alpha subunit was rapidly cleaved to a 24-kDa fragment. However, in the presence of the purified gamma 2 subunit, trypsin cleavage of the G(o) alpha subunit was completely prevented. This demonstration of a direct association between the gamma and alpha subunits is particularly intriguing in light of the increasingly large number of known alpha, beta, and gamma subunits, which raises important questions regarding the assembly of these subunits into functionally distinct G proteins. Thus, a direct association between the gamma and alpha subunits, which exhibit the greatest structural diversity, may provide the basis for the selective assembly of these subunits into G proteins with functional diversity.  相似文献   

14.
PURPOSE: To evaluate the role of ionic interactions in the cell surface expression of integrins and the organization of F-actin. Understanding these interactions will allow the development of surfaces for prosthetic purposes that will promote the normal expression of adhesion proteins. METHODS: Hema (hydroxyethylmethacrylate) hydrogels were used to mimic the charges present on extracellular matrix proteins. The surfaces were modified by the addition of amines (N,N-dimethylaminoethylmethacrylate; NDAM) or carboxyl moieties (methacrylic acid). The effects of ionic interactions on cellular spreading and on the expression of proteins were examined by modification of the stoichiometrically defined amounts of positive and negative charges on the Hemas. Changes in intracellular pH and the distribution and localization of protein were monitored using fluorescent markers, spectrofluorometry, and confocal laser scanning microscopy, respectively. The immunohistochemical studies were confirmed by flow cytometric analysis. RESULTS: The data indicate that although cells adhered to all the surfaces, the number of cells possessing adhesion receptors is significantly greater on surfaces with amine functionalities. Cell seeding and plating efficiency after 2 hours were identical on all surfaces. The intracellular pH of epithelial cells grown on surfaces containing NDAM, a tertiary amine, was higher than that of cells grown on Hemas containing only methacrylic acid. Lamellipodial extensions and an extensive actin network were present on surfaces containing 5% NDAM. The alpha 6 subunit was localized along the lateral cell membranes. The alpha 2 and 3 subunits were present along cell membranes and at lamellipodial extensions. Cells cultured on surfaces containing only methacrylic acid did not spread. Actin filaments were not detected, and alpha 6 was negligible on these surfaces. CONCLUSIONS: This is a novel approach to understanding cell-substrate interactions, and one that allows quantitative evaluation of the response of cells to defined surfaces. The organization of F-actin is altered by the substrates containing only carboxyl moieties. The distribution of integrin subunits is also altered by the substrate. These results indicate that epithelial cell spreading and protein expression may be regulated by ionic interactions.  相似文献   

15.
Poly(ethylene oxide) (PEO) polymer, in linear and star form, was covalently grafted to silicon surfaces, and the surfaces were tested for their ability to adsorb proteins. Linear PEG of molecular weight 3400, 10 000, and 20 000 g/mol and star PEO molecules were coupled via their terminal hydroxyl groups activated by tresyl chloride to aminosilane-treated silicon wafers. The amount of PEO coupled to the surface was varied by changing the concentration of the tresyl-PEO solution. The dry PEO thickness on the surface was measured using X-ray photoelectron spectroscopy (XPS) and ellipsometry, from which the grafting density was calculated. The PEO surfaces were exposed to solutions of each of three proteins: cytochrome-c, albumin, and fibronectin. The degree of adsorption of each protein was determined by XPS and ellipsometry and recorded as a function of PEO grafting density. All three proteins were found to reach zero adsorption at the highest grafting densities on all three PEG surfaces, which for all three PEG surfaces was a PEO content of 100 +/- 10 ng/cm2. On both star PEO surfaces, albumin and fibronectin decreased to zero adsorption at intermediate values of grafting density, whereas cytochrome-c continued to adsorb at all grafting densities, although with a decreasing trend. A physical model of the surface helped explain these protein adsorption results in terms of the spacing and degree of overlap of grafted PEO chains.  相似文献   

16.
Protein regulators of G protein signaling (RGS proteins) were discovered as negative regulators of heterotrimeric G protein-mediated signal transduction in yeast and worms. Experiments with purified recombinant proteins in vitro have established that RGS proteins accelerate the GTPase activity of certain G protein alpha subunits (the reaction responsible for their deactivation); they can also act as effector antagonists. We demonstrate herein that either of two such RGS proteins, RGS4 or GAIP, attenuated signal transduction mediated by endogenous receptors, G proteins, and effectors when stably expressed as tagged proteins in transfected mammalian cells. The pattern of selectivity observed in vivo was similar to that seen in vitro. RGS4 and GAIP both attenuated Gi-mediated inhibition of cAMP synthesis. RGS4 was more effective than GAIP in blocking Gq-mediated activation of phospholipase Cbeta.  相似文献   

17.
A family of GTP-binding regulatory proteins (G proteins) transduces signals across the plasma membrane from a large number of receptors to a smaller number of effectors. Recent studies indicate that a series of post-translational modifications are required for their association with the plasma membrane and for their function. In the case of the G protein gamma subunits, the post-translational modifications include the prenylation of a cysteine residue within a carboxyl-terminal CAAX motif. Although prenylation has been shown to involve the addition of either a C15 farnesyl or a C20 geranylgeranyl group to proteins, the structural requirements and functional consequences of adding different types of prenyl groups to various members of the gamma subunit family have not been examined. In the present study, we have employed the baculovirus expression system to study the structural requirements for attaching different types of prenyl groups to various members of the gamma subunit family. We show that the gamma 2 subunit is modified by a C20 geranylgeranyl group, consistent with the presence of a geranylgeranylation target sequence in this protein. However, we found that the gamma 1 and mutant gamma 2(Ser-71) subunits are modified by both C15 farnesyl and C20 geranylgeranyl groups, despite the presence of an accepted farnesylation target sequence in both of these proteins. Using chimeras of the gamma 1 and gamma 2 subunits, we provide evidence indicating that structural elements upstream of the carboxyl-terminal CAAX motif play a role in the recognition of members of the gamma subunit family by the appropriate insect and mammalian prenyltransferases.  相似文献   

18.
G-protein-coupled receptors (GPCRs) transduce the signals for a wide range of hormonal and sensory stimuli by activating a heterotrimeric guanine nucleotide-binding protein (G protein). The analysis of loss-of-function and constitutively active receptor mutants has helped to reveal the functional properties of GPCRs and their role in human diseases. Here we describe the identification of a new class of mutants, dominant-negative mutants, for the yeast G-protein-coupled alpha-factor receptor (Ste2p). Sixteen dominant-negative receptor mutants were isolated based on their ability to inhibit the response to mating pheromone in cells that also express wild-type receptors. Detailed analysis of two of the strongest mutant receptors showed that, unlike other GPCR interfering mutants, they were properly localized at the plasma membrane and did not alter the stability or localization of wild-type receptors. Furthermore, their dominant-negative effect was inversely proportional to the relative amount of wild-type receptors and was reversed by overexpressing the G-protein subunits, suggesting that these mutants compete with the wild-type receptors for the G protein. Interestingly, the dominant-negative mutations are all located at the extracellular ends of the transmembrane segments, defining a novel region of the receptor that is important for receptor signaling. Altogether, our results identify residues of the alpha-factor receptor specifically involved in ligand binding and receptor activation and define a new mechanism by which GPCRs can be inactivated that has important implications for the evaluation of receptor mutations in other G-protein-coupled receptors.  相似文献   

19.
G protein-coupled receptor activation leads to the membrane recruitment and activation of G protein-coupled receptor kinases, which phosphorylate receptors and lead to their inactivation. We have identified a novel G protein-coupled receptor kinase-interacting protein, GIT1, that is a GTPase-activating protein (GAP) for the ADP ribosylation factor (ARF) family of small GTP-binding proteins. Overexpression of GIT1 leads to reduced beta2-adrenergic receptor signaling and increased receptor phosphorylation, which result from reduced receptor internalization and resensitization. These cellular effects of GIT1 require its intact ARF GAP activity and do not reflect regulation of GRK kinase activity. These results suggest an essential role for ARF proteins in regulating beta2-adrenergic receptor endocytosis. Moreover, they provide a mechanism for integration of receptor activation and endocytosis through regulation of ARF protein activation by GRK-mediated recruitment of the GIT1 ARF GAP to the plasma membrane.  相似文献   

20.
Gastrin/CCKB G protein-coupled receptors have been shown to mediate proliferative effects of their endogenous ligands. In the present study, we examined the signal transduction mechanisms linked to the G/CCKB receptor occupancy. We report here that gastrin stimulates MAP kinase activation in a dose- and time-dependent manner, a pathway known to play a key role in cell proliferation. We also characterized the molecular events, upstream of p21-Ras, that may link the MAP kinase pathway to G/CCKB receptors. Gastrin induced a rapid and transient increase in tyrosine phosphorylation of several proteins including the 2 isoforms (46 and 52 kDa) of the adaptor protein Shc. Phosphorylated Shc subsequently associated with a complex that includes Grb2 and the p21-Ras activator, Sos. Our results also indicate that Sos becomes phosphorylated in response to gastrin as shown by a reduction in electrophoretic mobility of the protein. Tyrosine phosphorylation of Shc and subsequent complex formation with Grb2 and Sos appear to be a common mechanism by which tyrosine kinase receptors and the G/CCKB G protein-coupled receptor stimulate the Ras-dependent MAP kinase pathway.  相似文献   

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