Showing papers on "GTP-Binding Protein alpha Subunits published in 2002"
TL;DR: This review focuses on the coupling specificity of the Galpha and Gbetagamma subunits of pertussis toxin-sensitive G(i/o) proteins that mediate diverse signaling pathways, including regulation of ion channels and other effectors.
173 citations
TL;DR: It is demonstrated that G(alpha)(i) is not only a donor of G(beta)(gamma) but also regulates GIRK gating, and is regulated, in distinct ways, by both arms of the G protein.
152 citations
TL;DR: Translocation of G(alpha)(s)-GFP fusion protein induced by isoproterenol suggested that the internalization of G-Alpha)(s) might play a role in signal transduction by interacting with effector molecules and cytoskeletal elements at multiple cellular sites.
Abstract: To study behavior of activated G(alpha)(s) in living cells, green fluorescent protein (GFP) was inserted within the internal amino acid sequence of G(alpha)(s) to generate a G(alpha)(s)-GFP fusion protein. The fusion protein maintained a bright green fluorescence and was identified by immunoblotting with antibodies against G(alpha)(s) or GFP. The cellular distribution of G(alpha)(s)-GFP was similar to that of endogenous G(alpha)(s). G(alpha)(s)-GFP was tightly coupled to the beta adrenergic receptor to activate the G(alpha)(s) effector, adenylyl cyclase. Activation of G(alpha)(s)-GFP by cholera toxin caused a gradual displacement of the fusion protein from the plasma membrane throughout the cytoplasm in living cells. Unlike the slow release of G(alpha)(s)-GFP from the membrane induced by cholera toxin, the beta-adrenergic agonist isoproterenol caused a rapid partial release of the fusion protein into the cytoplasm. At 1 min after treatment with isoproterenol, the extent of G(alpha)(s)-GFP release from plasma membrane sites was maximal; however, insertion of G(alpha)(s)-GFP at other membrane sites occurred during the same time period. Translocation of G(alpha)(s)-GFP fusion protein induced by isoproterenol suggested that the internalization of G(alpha)(s) might play a role in signal transduction by interacting with effector molecules and cytoskeletal elements at multiple cellular sites.
119 citations
TL;DR: The findings suggest that alpha(1)AR-induced hypertrophic responses are mediated in part by a Galpha(12/13)-Rho-JNK pathway, in parts by a G(q/11)-JNK pathways that is Rho independent, and in partBy a Gbetagamma pathway that is JNK independent.
Abstract: In neonatal cardiomyocytes, activation of the G(q)-coupled alpha(1)-adrenergic receptor (alpha(1)AR) induces hypertrophy by activating mitogen-activated protein kinases, including c-Jun NH(2)-terminal kinase (JNK). Here, we show that JNK activation is essential for alpha(1)AR-induced hypertrophy, in that alpha(1)AR-induced hypertrophic responses, such as reorganization of the actin cytoskeleton and increased protein synthesis, could be blocked by expressing the JNK-binding domain of JNK-interacting protein-1, a specific inhibitor of JNK. We also identified the classes and subunits of G proteins that mediate alpha(1)AR-induced JNK activation and hypertrophic responses by generating several recombinant adenoviruses that express polypeptides capable of inhibiting the function of specific G-protein subunits. alpha(1)AR-induced JNK activation was inhibited by the expression of carboxyl terminal regions of Galpha(q), Galpha(12), and Galpha(13). JNK activation was also inhibited by the Galpha(q/11)- or Galpha(12/13)-specific regulator of G-protein signaling (RGS) domains and by C3 toxin but was not affected by treatment with pertussis toxin or by expression of the carboxyl terminal region of G protein-coupled receptor kinase 2, a polypeptide that sequesters Gbetagamma. alpha(1)AR-induced hypertrophic responses were inhibited by Galpha(q/11)- and Galpha(12/13)-specific RGS domains, C3 toxin, and the carboxyl terminal region of G protein-coupled receptor kinase 2 but not by pertussis toxin. Activation of Rho was inhibited by carboxyl terminal regions of Galpha(12) and Galpha(13) but not by Galpha(q). Our findings suggest that alpha(1)AR-induced hypertrophic responses are mediated in part by a Galpha(12/13)-Rho-JNK pathway, in part by a G(q/11)-JNK pathway that is Rho independent, and in part by a Gbetagamma pathway that is JNK independent.
100 citations
TL;DR: The data indicate that hMOR couples more efficiently to Gi1α than Gi2α and that the coupling efficacy is clearly agonist‐dependent, as well as indicating that ERK‐MAPK phosphorylation is initiated via coupling to endogenous G proteins only.
Abstract: As preferential coupling of opioid receptor to various inhibitory Galpha subunits is still under debate, we have investigated the selectivity of the human mu opioid receptor fused to a pertussis toxin insensitive C351I Gi1 alpha or C352I Gi2 alpha in stably transfected HEK 293 cells. Overall agonist binding affinities were increased for both fusion constructs when compared to the wild type receptor. [35 S]GTPgammaS binding was performed on pertussis toxin treated cells to monitor coupling efficiency of the fusion constructs. Upon agonist addition hMOR-C351I Gi1 a exhibited an activation profile similar to the non-fused receptor while hMOR-C352I Gi2 alpha was poorly activated. Interestingly no correlation could be drawn between agonist binding affinity and efficacy. Upon agonist addition, forskolin-stimulated cAMP production, as measured using a reporter gene assay, was inhibited by signals transduced via the fused Gi1 alpha and Gi2 alpha mainly. In contrast both fusion constructs were able to initiate ERK-MAPK phosphorylation via coupling to endogenous G proteins only. In conclusion our data indicate that hMOR couples more efficiently to Gi1 alpha than Gi2 alpha and that the coupling efficacy is clearly agonist-dependent.
48 citations
TL;DR: Various fragments were shown to be liberated by the action of prohormone convertases, in accordance with the concept that these peptides represent the mediators of at least part of the yet unidentified physiological functions of the chromogranins.
Abstract: A number of peptides derived from the large chromogranin precursor proteins have been identified in neuroendocrine tissues and several of them have been attributed with functional data. In addition, various fragments were shown to be liberated by the action of prohormone convertases. This is in accordance with the concept that these peptides represent the mediators of at least part of the yet unidentified physiological functions of the chromogranins.
32 citations
TL;DR: Flow cytometry permits quantiatitive and real-time assessments of protein-protein interactions in complex membrane environments and suggests betagamma contributed essentially all of the binding energy for alpha(i1) interaction with the membrane.
Abstract: The dynamics of G protein heterotrimer complex formation and disassembly in response to nucleotide binding and receptor activation govern the rate of responses to external stimuli. We use a novel flow cytometry approach to study the effects of lipid modification, isoform specificity, lipid environment, and receptor stimulation on the affinity and kinetics of G protein subunit binding. Fluorescein-labeled myristoylated Galpha(i1) (F-alpha(i1)) was used as the ligand bound to Gbetagamma in competition binding studies with differently modified Galpha subunit isoforms. In detergent solutions, the binding affinity of Galpha(i) to betagamma was 2 orders of magnitude higher than for Galpha(o) and Galpha(s) (IC50 of 0.2 nM vs 17 and 27 nM, respectively), while in reconstituted bovine brain lipid vesicles, binding was slightly weaker. The effects of receptor on the G protein complex were assessed in alpha(2A)AR receptor expressing CHO cell membranes into which purified betagamma subunits and F-alpha(i1) were reconstituted. These cell membrane studies led to the following observations: (1) binding of alpha subunit to the betagamma was not enhanced by receptor in the presence or absence of agonist, indicating that betagamma contributed essentially all of the binding energy for alpha(i1) interaction with the membrane; (2) activation of the receptor facilitated GTPgammaS-stimulated detachment of F-alpha(i1) from betagamma and the membrane. Thus flow cytometry permits quantiatitive and real-time assessments of protein-protein interactions in complex membrane environments.
29 citations
TL;DR: The data suggest that a mechanism of the GDI activity of GPR-proteins is different from that of GDIs for monomeric GTPases and from theGDI-like activity ofG(betagamma) subunits.
Abstract: Proteins containing G-protein regulatory (GPR) motifs represent a novel family of guanine nucleotide dissociation inhibitors (GDIs) for G(alpha) subunits from the Gi family. They selectively interact with the GDP-bound conformation of Gi(alpha) and transducin-alpha (Gt(alpha)), but not with Gs(alpha). A series of chimeric proteins between Gi(alpha)(1) and Gs(alpha) has been constructed to investigate GPR-contact sites on G(alpha) subunits and the mechanism of GPR-protein GDI activity. Analysis of the interaction of two GPR-proteins-AGS3GPR and Pcp2-with the chimeric G(alpha) subunits demonstrated that the GPR-Gi(alpha)(1) interface involves the Gi(alpha)(1) switch regions and Gi(alpha)(1)-144-151, a site within the helical domain. Residues within Gi(alpha)(1)-144-151 form conformation-sensitive contacts with switch III, and may directly interact with a GPR-protein or form a GPR-binding surface jointly with switch III. The helical domain site is critical to the ability of GPR-proteins to act as GDIs. Our data suggest that a mechanism of the GDI activity of GPR-proteins is different from that of GDIs for monomeric GTPases and from the GDI-like activity of G(betagamma) subunits. The GPR-proteins are likely to block a GDP-escape route on G(alpha) subunits.
20 citations
TL;DR: This work has identified the Gαolf subunit in rat pancreatic β-cells and investigated the downstream effectors and the biological functions of this G protein in HEK-293T cells and the insulin-dependent response of these cells.
Abstract: Because we recently identified the G(alpha)olf subunit in rat pancreatic beta-cells, we investigated the downstream effectors and the biological functions of this G protein in HEK-293T cells and the insulin-secreting mouse betaTC-3 cell line. With the use of transient transfection of HEK-293T cells with constitutively activated G(alpha)olf (G(alpha)olfQ214L, i.e., AG(alpha)olf), together with expression vectors encoding the adenylyl cyclase (AC) isoforms (AC-I to -VIII and soluble AC), compared with cotransfections using AG(alphas) (G(alphas)R201C), we observed that AG(alpha)olf preferentially activates AC-I and -VIII, which are also expressed in beta-cells. Stable overexpression of wild-type or AG(alpha)olf in betaTC-3 cells resulted in partial attenuation of insulin secretion and biosynthesis, suggesting that chronic activation of the G(alpha)olf-signaling pathway is associated with beta-cell desensitization. In agreement, transfected betaTC-3 cells present a decreased insulin content with respect to parental cells, whereas the proinsulin convertases PC-1 and PC-2 were unaffected. Furthermore, betaTC-3-AG(alpha)olf cells are resistant to serum starvation-induced apoptosis. Our findings suggest that G(alpha)olf is involved in insulin status, cell survival, and regeneration of the insulin-secreting beta-cells during development and diabetes.
18 citations
TL;DR: The creation of transgenic mice with an inducible, tissue-targeted expression of a constitutively active mutant form (Q227L) of Gαs is reported, which results in mice expressing activated G αs in fat tissue, liver, an...
Abstract: We report the creation of transgenic mice with an inducible, tissue-targeted expression of a constitutively active mutant form (Q227L) of G(alpha)(s). Mice expressing activated G(alpha)(s) in fat tissue, liver, and skeletal muscle displayed normal body mass and blunted glucose metabolism. cAMP accumulation in adipose tissue was increased in the basal state, but far less than would be expected. Marked adaptation to elevated cAMP levels occurred, leading to an increase in total cAMP-specific phosphodiesterase activity, a 50% decline in cAMP-dependent protein kinase (protein kinase A) activity, and an increased expression of G(alpha)(i2). The reduction in kinase activity coincided with >50% increase in the expression of RIalpha and RIIalpha regulatory subunits of protein kinase A, with no change in the amount of catalytic subunit. These data demonstrate the existence of adaptive responses of protein kinase A, phosphodiesterase, and G(alpha)(i2) in tissues expressing constitutively active G(alpha)(s) that may act to rectify the impact of increased cAMP accumulation.
13 citations
TL;DR: AC activation is explained by a model in which there is interaction of the beta(2)AR and AC, preventing tethered G(i)alpha(2), a preferentially G(s)-coupled receptor, from interacting with the inhibitory G( i)alpha site of AC.
TL;DR: The genomic sequences of the NtGA loci in tobacco and its ancestral diploid species were concluded to be homoeologous and assigned to the S and T genomes, respectively, and more than 300 mutations including insertions-deletions (indels) and nucleotide substitutions were found in the intron regions between the NTGA1 and Nt GA2 loci.
Abstract: A heterotrimeric GTP-binding protein (G protein) plays a number of important roles in the signal- transduction pathways of eukaryotic cells. The allotetraploid tobacco genome has two α-subunit gene...
TL;DR: Since patients with PHP have resistance to other hormones in addition to PTH, such as TSH and gonadotropins, which work via cAMP, the molecular defect could not reside at the receptor level, but rather must be at a more distal component of the signaling pathway.
Abstract: In 1942, Albright et al. described pseudohypoparathyroidism (PHP), the first hormone-resistant disorder. They investigated three patients with the clinical picture of hypoparathyroidism who did not respond to parathyroid (PTH) extract, and postulated that \"there is a deficiency in or interference with some hypothetical substance with which the parathyroid hormone reacts\". The finding that PTH stimulates renal adenylate cyclase to increase cAMP synthesis led to the experiment of Chase et al., who showed that patients with PHP did not increase urinary cAMP in response to PTH administration. They concluded that \"the metabolic defect can be accounted for by a lack of or defective form of PTH-sensitive adenylate cyclase in bone and kidney\". Since patients with PHP have resistance to other hormones in addition to PTH, such as TSH and gonadotropins, which work via cAMP, the molecular defect could not reside at the receptor level, but rather must be at a more distal component of the signaling pathway. The G protein which couples hormone receptor to stimulation of adenylate cyclase (Gs) was found to harbor the molecular defect'. G proteins (a family of about 20 members) are composed of three peptides; α, β and γ. The α subunit binds guanine nucleotides, and interacts with the specific receptor and effector . A biochemical complementation assay showed that the erythrocytes of patients with PHP had 50% G sa activity compared to those of normal individuals.