Arf GAPs and membrane traffic
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Abstract:
The selective transfer of material between membrane-delimited organelles is mediated by protein-coated vesicles. In many instances, formation of membrane trafficking intermediates is regulated by the GTP-binding protein Arf. Binding and hydrolysis of GTP by Arf was originally linked to the assembly and disassembly of vesicle coats. Arf GTPase-activating proteins (GAPs), a family of proteins that induce hydrolysis of GTP bound to Arf, were therefore proposed to regulate the disassembly and dissociation of vesicle coats. Following the molecular identification of Arf GAPs, the roles for GAPs and GTP hydrolysis have been directly examined. GAPs have been found to bind cargo and known coat proteins as well as directly contribute to vesicle formation, which is consistent with the idea that GAPs function as subunits of coat proteins rather than simply Arf inactivators. In addition, GTP hydrolysis induced by GAPs occurs largely before vesicle formation and is required for sorting. These results are the primary basis for modifications to the classical model for the function of Arf in transport vesicle formation, including a recent proposal that Arf has a proofreading, rather than a structural, role.Keywords:
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Background and Design. - Heterotrimeric guanine nucleotide binding protein (G proteins) play a central role in regulation of signal transmission in the cell. G proteins which are localized in the inner surface of the cell membrane consist of α-, β- and γ- subunits. The α-subunit which binds guanine nucleotides (GTP and GDP) contains intrinsic GTPase activity. G proteins are divided into four families based on their α-subunits which confer their specifity: Gαs, Gαi, Gαq and Gα12. This study was designed to measure GTP binding activity and G protein expression in rat brain. Membrane extracts were prepared from whole brain and brain cortex. GTP binding activity in crude membrane fractions (P30) and membrane extracts (S142) was measured using [35S] GTPγS, the non-hydrolyzable analogue of GTP. Conclusion. - We observed that [35S] GTPγS binding increased with time and with increasing amounts of membrane proteins. We also demonstrated that GTPγS binding was strongly magnesium dependent and was maximum at 60mM MgCl2 concentration. The presence of G protein α and βγ subunits in whole brain and of Gαo and Gαi’ in brain cortex was shown by Western blot analysis. *Anahtar Kelimeler: G proteinler, GTP baglama, beyin *Key Words: G proteins, GTP binding, brain
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The publishers wish to apologize for errors that appeared in the above article published in J. Neurochem.78, pp. 325–338. Here we show Tables 1 and 2 and a section of text from pp. 331–332 as they should have appeared (the corrected text is set in bold). To ensure sensitive detection of GPCR-stimulated GTPγS binding to Gαolf and GsαS, we determined GTPγS binding in the presence of a large molar excess of GDP relative to GTPγS (see Materials and methods) (Wenzel-Seifert and Seifert 2000). In membranes expressing β2AR-Gαolf, the agonist-stimulated t1/2 of GTPγS binding (6.2 ± 2.1 min) was about half of that of the corresponding t1/2 of GTPγS binding to β2AR-GsαS (11.2 ± 2.4 min; Fig. 4). We also noticed that t1/2 of basal GTPγS binding in membranes expressing β2AR-Gαolf (26.1 ± 5.9 min) was lower than in membranes expressing β2AR-GsαS (48.7± 6.8 min), indicating that in membranes expressing β2AR-Gαolf some agonist-independent GTPγS binding occurred. In fact, the inverse agonist ICI exhibited a significant inhibitory effect on basal GTPγS binding in membranes expressing β2AR-Gαolf (Fig. 5). These data support the hypothesis that GTPγS binds to Gαolf more rapidly than to GsαS and that the agonist-free β2AR promotes GTPγS binding to Gαolf more efficiently than to GsαS.
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Regulator of G protein Signaling, or RGS, proteins serve an important regulatory role in signaling mediated by G protein-coupled receptors (GPCRs). They all share a common RGS domain that directly interacts with active, GTP-bound Gα subunits of heterotrimeric G proteins. RGS proteins stabilize the transition state for GTP hydrolysis on Gα and thus induce a conformational change in the Gα subunit that accelerates GTP hydrolysis, thereby effectively turning off signaling cascades mediated by GPCRs. This GTPase accelerating protein (GAP) activity is the canonical mechanism of action for RGS proteins, although many also possess additional functions and domains. RGS proteins are divided into four families, R4, R7, R12 and RZ based on sequence homology, domain structure as well as specificity towards Gα subunits. For reviews on RGS proteins and their potential as therapeutic targets, see e.g. [225, 529, 578, 583, 584, 742, 753, 444, 10].
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Many Regulators of G proteinSignaling (RGS) proteins accelerate the intrinsic GTPase activity of Giα and Gqα-subunits [i.e., behave as GTPase-activating proteins (GAPs)] and several act as Gqα-effector antagonists. RGS3, a structurally distinct RGS member with a unique N-terminal domain and a C-terminal RGS domain, and an N-terminally truncated version of RGS3 (RGS3CT) both stimulated the GTPase activity of Giα (except Gzα) and Gqα but not that of Gsα or G12α. RGS3 and RGS3CT had Gqα GAP activity similar to that of RGS4. RGS3 impaired signaling through Gq-linked receptors, although RGS3CT invariably inhibited better than did full-length RGS3. RGS3 potently inhibited GqαQ209L- and G11αQ209l-mediated activation of a cAMP-response element-binding protein reporter gene and GqαQ209L induced inositol phosphate production, suggesting that RGS3 efficiently blocks Gqα from activating its downstream effector phospholipase C-β. Whereas RGS2 and to a lesser extent RGS10 also inhibited signaling by these GTPase-deficient G proteins, other RGS proteins including RGS4 did not. Mutation of residues in RGS3 similar to those required for RGS4 Giα GAP activity, as well as several residues N terminal to its RGS domain impaired RGS3 function. A greater percentage of RGS3CT localized at the cell membrane than the full-length version, potentially explaining why RGS3CT blocked signaling better than did full-length RGS3. Thus, RGS3 can impair Gi- (but not Gz-) and Gq-mediated signaling in hematopoietic and other cell types by acting as a GAP for Giα and Gqα subfamily members and as a potent Gqα subfamily effector antagonist.
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▪ Abstract GTPase-activating proteins (GAPs) regulate heterotrimeric G proteins by increasing the rates at which their α subunits hydrolyze bound GTP and thus return to the inactive state. G protein GAPs act allosterically on Gα subunits, in contrast to GAPs for the Ras-like monomeric GTP-binding proteins. Although they do not contribute directly to the chemistry of GTP hydrolysis, G protein GAPs can accelerate hydrolysis >2000-fold. G protein GAPs include both effector proteins (phospholipase C-β, p115RhoGEF) and a growing family of regulators of G protein signaling (RGS proteins) that are found throughout the animal and fungal kingdoms. GAP activity can sharpen the termination of a signal upon removal of stimulus, attenuate a signal either as a feedback inhibitor or in response to a second input, promote regulatory association of other proteins, or redirect signaling within a G protein signaling network. GAPs are regulated by various controls of their cellular concentrations, by complex interactions with Gβγ or with Gβ5 through an endogenous Gγ-like domain, and by interaction with multiple other proteins.
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