Activation of G12/G13 results in shape change and Rho/Rho-kinase-mediated myosin light chain phosphorylation in mouse platelets.
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Platelets respond to various stimuli with rapid changes in shape followed by aggregation and secretion of their granule contents. Platelets lacking the alpha-subunit of the heterotrimeric G protein Gq do not aggregate and degranulate but still undergo shape change after activation through thromboxane-A2 (TXA2) or thrombin receptors. In contrast to thrombin, the TXA2 mimetic U46619 led to the selective activation of G12 and G13 in Galphaq-deficient platelets indicating that these G proteins mediate TXA2 receptor-induced shape change. TXA2 receptor-mediated activation of G12/G13 resulted in tyrosine phosphorylation of pp72(syk) and stimulation of pp60(c-src) as well as in phosphorylation of myosin light chain (MLC) in Galphaq-deficient platelets. Both MLC phosphorylation and shape change induced through G12/G13 in the absence of Galphaq were inhibited by the C3 exoenzyme from Clostridium botulinum, by the Rho-kinase inhibitor Y-27632 and by cAMP-analogue Sp-5,6-DCl-cBIMPS. These data indicate that G12/G13 couple receptors to tyrosine kinases as well as to the Rho/Rho-kinase-mediated regulation of MLC phosphorylation. We provide evidence that G12/G13-mediated Rho/Rho-kinase-dependent regulation of MLC phosphorylation participates in receptor-induced platelet shape change. (+info)
Tyrosine kinase Pyk2 mediates G-protein-coupled receptor regulation of the Ewing sarcoma RNA-binding protein EWS.
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Ewing family tumors result from the effects of chromosomal translocations that fuse the Ewing sarcoma (EWS) gene to various genes encoding transcription factors. The resulting chimeric EWS fusion proteins are transcriptional activators with transforming potential that have received much study. By contrast, the cellular function of somatic EWS remains obscure. EWS belongs to a family of RNA-binding proteins thought to play role in RNA synthesis or processing. Here, we show that EWS interacts with Pyk2, a protein tyrosine kinase implicated in a variety of signal transduction processes. G-protein-coupled receptor signaling and other stimuli of Pyk2 kinase activity significantly block the interaction between EWS and Pyk2. Furthermore, as assessed by sucrose gradient centrifugation, EWS partitions with dense ribosome-containing fractions in a manner that is enhanced by signaling from the G-protein-coupled m1 muscarinic acetylcholine receptor (mAChR). We conclude that extranuclear EWS is a previously unrecognized target of G-protein-coupled receptor regulation. (+info)
Low- and high-level transgenic expression of beta2-adrenergic receptors differentially affect cardiac hypertrophy and function in Galphaq-overexpressing mice.
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Transgenic overexpression of Galphaq in the heart triggers events leading to a phenotype of eccentric hypertrophy, depressed ventricular function, marked expression of hypertrophy-associated genes, and depressed beta-adrenergic receptor (betaAR) function. The role of betaAR dysfunction in the development of this failure phenotype was delineated by transgenic coexpression of the carboxyl terminus of the betaAR kinase (betaARK), which acts to inhibit the kinase, or concomitant overexpression of the beta2AR at low (approximately 30-fold, Galphaq/beta2ARL), moderate (approximately 140-fold, Galphaq/beta2ARM), and high (approximately 1,000-fold, Galphaq/beta2ARH) levels above background betaAR density. Expression of the betaARK inhibitor had no effect on the phenotype, consistent with the lack of increased betaARK levels in Galphaq mice. In marked contrast, Galphaq/beta2ARL mice displayed rescue of hypertrophy and resting ventricular function and decreased cardiac expression of atrial natriuretic factor and alpha-skeletal actin mRNA. These effects occurred in the absence of any improvement in basal or agonist-stimulated adenylyl cyclase (AC) activities in crude cardiac membranes, although restoration of a compartmentalized beta2AR/AC signal cannot be excluded. Higher expression of receptors in Galphaq/beta2ARM mice resulted in salvage of AC activity, but hypertrophy, ventricular function, and expression of fetal genes were unaffected or worsened. With approximately 1,000-fold overexpression, the majority of Galphaq/beta2ARH mice died with cardiomegaly at 5 weeks. Thus, although it appears that excessive, uncontrolled, or generalized augmentation of betaAR signaling is deleterious in heart failure, selective enhancement by overexpressing the beta2AR subtype to limited levels restores not only ventricular function but also reverses cardiac hypertrophy. (+info)
A lobster phospholipase C-beta that associates with G-proteins in response to odorants.
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A cDNA clone encoding a protein of 1116 amino acids with significant homology to beta-isoforms of phospholipase C was isolated from lobster olfactory organ cDNA libraries and named lobPLCbeta. This cDNA hybridized predominantly to a 9 kb transcript in RNA from olfactory organ, pereiopod, brain, and eye-eyestalk and to several smaller minor transcripts only in eye-eyestalk. An antiserum raised to the C terminus of lobPLCbeta detected immunoreactivity in a single 130 kDa band in olfactory aesthetasc hairs, olfactory organ, pereiopod, dactyl, and brain. In eye-eyestalk this 130 kDa band was abundant, and minor bands of 100, 79, and 57 kDa also were detected. In cross sections of the aesthetasc hairs, immunoreactivity was detected in the outer dendritic segments of the olfactory receptor neurons, the site of olfactory transduction. A complex odorant caused lobPLCbeta immunoreactivity to increase in membrane fractions and decrease in soluble fractions of homogenates of aesthetasc hairs. The odorant also increased the amount of lobPLCbeta in immunoprecipitates of Galphaq and Gbeta from homogenates of aesthetasc hairs. These results support the conclusion that lobPLCbeta mediates olfactory transduction. (+info)
Amino-terminal cysteine residues of RGS16 are required for palmitoylation and modulation of Gi- and Gq-mediated signaling.
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RGS proteins (Regulators of G protein Signaling) are a recently discovered family of proteins that accelerate the GTPase activity of heterotrimeric G protein alpha subunits of the i, q, and 12 classes. The proteins share a homologous core domain but have divergent amino-terminal sequences that are the site of palmitoylation for RGS-GAIP and RGS4. We investigated the function of palmitoylation for RGS16, which shares conserved amino-terminal cysteines with RGS4 and RGS5. Mutation of cysteine residues at residues 2 and 12 blocked the incorporation of [3H]palmitate into RGS16 in metabolic labeling studies of transfected cells or into purified RGS proteins in a cell-free palmitoylation assay. The purified RGS16 proteins with the cysteine mutations were still able to act as GTPase-activating protein for Gialpha. Inhibition or a decrease in palmitoylation did not significantly change the amount of protein that was membrane-associated. However, palmitoylation-defective RGS16 mutants demonstrated impaired ability to inhibit both Gi- and Gq-linked signaling pathways when expressed in HEK293T cells. These findings suggest that the amino-terminal region of RGS16 may affect the affinity of these proteins for Galpha subunits in vivo or that palmitoylation localizes the RGS protein in close proximity to Galpha subunits on cellular membranes. (+info)
Phospholipase C-beta1 directly accelerates GTP hydrolysis by Galphaq and acceleration is inhibited by Gbeta gamma subunits.
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Phospholipase C-beta, the principal effector protein regulated by Galphaq, has been shown to increase the agonist-stimulated, steady-state GTPase activity of Gq in proteoliposomes that contain both heterotrimeric Gq and m1 muscarinic receptor. We now use a moderately stable complex of R183C Galphaq bound to GTP to show that PLC-beta1 acts directly as a GTPase-activating protein (GAP) for isolated Galphaq in a membrane-free system. PLC-beta1 accelerated the hydrolysis of GalphaqR183C.GTP up to 20-fold. The Km was 1.5 nM, which is similar both to the EC50 with which R183C and wild type Galphaq activate PLC-beta1 and to the EC50 with which PLC-beta1 acts as a Gq GAP in the vesicle-based assay. The Galphaq GAP activity of RGS4 can also be quantitated by this assay; it accelerated hydrolysis of bound GTP about 100-fold. The Gq GAP activities of both PLC-beta1 and RGS4 are blocked by Gbeta gamma subunits, probably by a competitive mechanism. These data suggest either that the Gbeta gamma subunits are not continuously required for receptor-catalyzed GDP/GTP exchange during steady-state GTP hydrolysis or that GAPs, either PLC-beta or RGS proteins, can substitute for Gbeta gamma in this set of reactions. (+info)
Rapid GTP binding and hydrolysis by G(q) promoted by receptor and GTPase-activating proteins.
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Receptor-promoted GTP binding and GTPase-activating protein (GAP)-promoted GTP hydrolysis determine the onset and termination of G protein signaling; they coordinately control signal amplitude. The mechanisms whereby cells independently regulate signal kinetics and signal amplitude are therefore central to understanding G protein function. We have used quench-flow kinetic methods to measure the rates of the individual reactions of the agonist-stimulated GTPase cycle for G(q) during steady-state signaling. G(q) and m1 muscarinic cholinergic receptor were co-reconstituted into proteoliposomes with one of two GAPs: phospholipase C (PLC)-beta1, the major G(q)-regulated effector protein, and RGS4, a GAP commonly thought to be an inhibitor of G(q) signaling. In this system, the rate constant for GAP-stimulated hydrolysis of Galpha(q)-bound GTP at 30 degrees C was 9-12 s(-1) for PLC-beta1 and 22-27 s(-1) for RGS4. These rates are 1,000- to 2,000-fold faster than in the absence of a GAP and far faster than measured previously. G(q) can thus hydrolyze bound GTP with deactivation half-times of 25-75 ms at 30 degrees C, commensurate with physiological rates of signal termination. GDP/GTP exchange, which reactivates G(q), was the principal rate-limiting step for the GTPase cycle and was also faster than previously thought. At physiological concentrations of GTP, exchange was limited by the rate of dissociation of GDP from the receptor-G(q) complex, with a maximal rate of 1.8 s(-1) at 30 degrees C. Comparison of activation and deactivation rates help explain how GDP/GTP exchange balance rapid GTP hydrolysis to maintain steady-state signal amplitude. (+info)
Transgenic replacement of type V adenylyl cyclase identifies a critical mechanism of beta-adrenergic receptor dysfunction in the G alpha q overexpressing mouse.
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Chronic activation of Gq coupled receptors, or overexpression of G alpha q, in cardiomyocytes results in hypertrophy, enhanced expression of fetal genes, decreased basal and beta-adrenergic receptor (beta AR) stimulated adenylyl cyclase (AC) activities, and depressed cardiac contractility in vivo. Among several abnormalities of the beta AR-Gs-AC pathway that occur in G alpha q overexpressing transgenic mice, we have investigated whether the observed approximately 45% decrease in type V AC expression and function compared to non-transgenic (NTG) is the basis of the above phenotype. Transgenic mice were generated that overexpressed by approximately 50% the rat type V AC in the heart using the alpha-myosin heavy chain promoter. These mice were mated with the G alpha q transgenics resulting in animals (ACV/G alpha q) that had restored levels of forskolin stimulated AC activities in cardiac membranes. In addition, basal cardiac AC activities were normalized in the ACV/G alpha q mice (NTG=23+/-4.4, G alpha q=14+/-3.6, ACV/G alpha q=29+/-5.3 pmol/min/mg) as were maximal isoproterenol stimulated activities (59+/-8.9, 34+/-4.6, 52+/-6.7 pmol/min/mg respectively). Cardiac contractility was also improved by ACV replacement, with increased fractional shortening (51+/-2%, 36+/-6%, 46+/-3% respectively). In contrast, hypertrophy and expression of hypertrophy associated fetal genes were not affected. Thus the observed decrease in type V AC that accompanies the development of the cardiac phenotype in the G alpha q model is the dominant mechanism of dysfunctional beta AR signalling and contractility. In contrast, the decrease in type V AC or beta AR signalling to cAMP is not the basis of the hypertrophic response. (+info)