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GTP-Binding Protein alpha Subunits

About: GTP-Binding Protein alpha Subunits is a research topic. Over the lifetime, 304 publications have been published within this topic receiving 19915 citations.


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Journal ArticleDOI
TL;DR: The cAMP-dependent protein kinase plays an intimate positive and negative role in regulating filamentous growth, and these findings may provide insight into the roles of PKA in mating, morphogenesis, and virulence in other yeasts and pathogenic fungi.
Abstract: In response to nitrogen limitation, diploid cells of Saccharomyces cerevisiae undergo a dimorphic transition to a filamentous growth form referred to as pseudohyphal differentiation (14, 19). This filamentous growth form represents a dramatic change in the cellular program in which the cells elongate, adopt a unipolar budding pattern, remain physically connected in chains, and invade the agar (14, 23). This alternative growth form may enable this nonmotile species to forage for nutrients under adverse conditions. Two signaling pathways that regulate yeast filamentous growth have been defined. The first involves components of the mitogen-activated protein (MAP) kinase pathway that also functions during mating in haploid cells (8, 28, 35). These components include the kinases Ste20, Ste11, Ste7, and Kss1 and the transcription factor Ste12. In addition, the transcription factor Tec1 forms heterodimers with Ste12 that regulate expression of Tec1 itself and additional targets, such as the cell surface flocculin Flo11 required for invasive and filamentous growth (3, 11, 31, 34). Early elements of the pheromone response pathway, including the pheromones, their receptors, and the coupled heterotrimeric G protein, are not expressed in diploid cells and are not required for filamentous differentiation (28). Instead, the MAP kinase pathway is activated by Cdc42, Ras2, and the 14-3-3 proteins Bmh1 and Bmh2 (38, 39, 49), possibly in response to the Sho1 osmosensing receptor (46). A second signaling pathway functions in parallel with the MAP kinase pathway to regulate pseudohyphal differentiation. This pathway involves a novel G protein-coupled receptor, Gpr1, which is required for both pseudohyphal differentiation (33) and, in conjunction with Ras2, vegetative growth (63). The Gpr1 ligand has not yet been identified. The Gpr1 receptor is coupled to a heterotrimeric G protein α subunit, Gpa2, which is also required for pseudohyphal differentiation and plays a role in nutrient sensing (26, 32). Early studies suggested Gpa2 might stimulate cyclic AMP (cAMP) production by adenylyl cyclase (41). Consistent with this, cAMP stimulates pseudohyphal differentiation and suppresses the filamentation defects of gpr1 and gpa2 mutant strains (26, 32, 33). A recent study has confirmed that Gpa2 regulates cAMP production by adenylyl cyclase in response to nutritional signals (7). Dominant activated Gpa2 mutants or cAMP suppresses the pseudohyphal defect of mutant strains lacking MAP kinase cascade components (32). In summary, a second signaling pathway comprised of the Gpr1 receptor, the Gpa2 Gα protein, and cAMP regulates pseudohyphal growth in parallel to and independently from the MAP kinase pathway. The target of cAMP in yeast is the cAMP-dependent protein kinase, protein kinase A (PKA). The yeast PKA kinase is similar to mammalian PKA and consists of a regulatory subunit encoded by a single gene, BCY1, and three catalytic subunits encoded by the TPK1, TPK2, and TPK3 genes (6, 57, 58). In both yeast and mammals, PKA in resting cells is an inactive tetramer composed of two regulatory subunits bound to two active subunits. In response to external signals that increase intracellular cAMP levels, cAMP binds to the regulatory subunit and triggers conformational changes that release the active catalytic subunits. Hydrolysis of cAMP by cAMP phosphodiesterases, the products of the PDE1 and PDE2 genes in yeast, restores PKA to the resting, inactive state (43, 54). The yeast cAMP-dependent protein kinase is required for vegetative growth (58). Triple mutants lacking the Tpk1, Tpk2, and Tpk3 catalytic subunits are inviable, whereas mutant strains expressing any one of the three Tpk subunits are all viable. These findings led to the model that the three PKA catalytic subunits are largely redundant for function. The PKA catalytic subunits share a conserved C-terminal kinase domain attached to unique N-terminal regions. Tpk1 and Tpk3 share 88% identity in the kinase domain, whereas Tpk2 is more divergent (77 and 75% identity with Tpk1 and -3, respectively). Several candidate PKA targets for vegetative growth have recently been identified. For example, the Msn2 and Msn4 transcription factors are regulated by PKA and repress expression of genes that regulate vegetative growth (4, 18, 56). The Rim15 protein kinase is also phosphorylated and inhibited by PKA and regulates entry into meiosis and stationary phase (48, 60). In parallel, studies of PKA constitutively activated by cAMP, bcy1 mutation, or activated Ras2 revealed roles in regulating stationary phase, meiosis, and sporulation (5, 59). Activation of PKA prevents glycogen accumulation, heat shock resistance, and survival during nutrient limitation, all hallmarks of entry into stationary phase. Similarly, activation of PKA inhibits sporulation. Thus, activation of PKA promotes vegetative growth in response to nutrients, whereas inactivation of PKA in response to nutrient limitation regulates sporulation and entry into stationary phase. Several observations suggest PKA might also regulate yeast pseudohyphal differentiation. The dominant active Ras2val19 mutant protein enhances filamentous growth (14), whereas overexpression of the cAMP phosphodiesterase Pde2 inhibits filamentation (62). In addition, exogenous cAMP enhances filamentous growth (32). Here we report that the cAMP-dependent protein kinase regulates yeast pseudohyphal differentiation. First, we show that mutation of the PKA regulatory subunit Bcy1 enhances filamentous growth. Second, we demonstrate that the PKA catalytic subunits play distinct roles in regulating filamentous growth: the Tpk2 subunit activates filamentous growth, whereas the Tpk1 and Tpk3 subunits primarily inhibit filamentous growth. The unique activating function of the Tpk2 subunit is linked to structural differences in the catalytic region of the kinase and not to differences in gene regulation or the unique amino-terminal region of the protein. Genetic epistasis experiments support a model in which Tpk2 functions downstream of the Gpr1 receptor and the Gα protein Gpa2. Importantly, activation of PKA by mutation of the Bcy1 regulatory subunit restores pseudohyphal growth in mutants lacking elements of the MAP kinase pathway, including ste12, tec1, and ste12 tec1 mutant strains. Thus, the MAP kinase and PKA pathways independently regulate filamentous growth. Further analysis reveals that the PKA pathway regulates the switch to unipolar budding and invasion, whereas the MAP kinase pathway is required for cell elongation and invasion. Finally, our studies define a role for the PKA pathway in activating pseudohyphal growth via transcriptional regulation of the cell surface flocculin Flo11 by the Flo8 transcription factor, and both Flo11 and Flo8 were previously shown to be required for pseudohyphal growth (27, 29, 31). Taken together, our studies reveal an intimate role for the cAMP-dependent kinase in the regulation of yeast dimorphism and suggest this role has been evolutionarily conserved in diverse yeast species and fungi, including pathogens of both plants and animals.

364 citations

Journal ArticleDOI
15 Jun 2001-Science
TL;DR: Results from loss of function and ectopic expression and activation of GPA1 indicate that this subunit is a positive modulator of cell division in plants.
Abstract: The alpha subunit of a prototypical heterotrimeric GTP-binding protein (G protein), which is encoded by a single gene (GPA1) in Arabidopsis, is a modulator of plant cell proliferation. gpa1 null mutants have reduced cell division in aerial tissues throughout development. Inducible overexpression of GPA1 in Arabidopsis confers inducible ectopic cell division. GPA1 overexpression in synchronized BY-2 cells causes premature advance of the nuclear cycle and the premature appearance of a division wall. Results from loss of function and ectopic expression and activation of GPA1 indicate that this subunit is a positive modulator of cell division in plants.

363 citations

Journal ArticleDOI
TL;DR: A thorough understanding of the molecular and physiological functions of Gβγ has significant implications for regulation of G protein-coupled receptor signaling pathways.
Abstract: G protein βγ subunits are central participants in G protein-coupled receptor signaling pathways. They interact with receptors, G protein α subunits and downstream targets to coordinate multiple, different GPCR functions. Much is known about the biology of Gβγ subunits but mysteries remain. Here, we will review what is known about general aspects of structure and function of Gβγ as well as discuss emerging mechanisms for regulation of Gβγ signaling. Recent data suggest that Gβγ is a potential therapeutic drug target. Thus, a thorough understanding of the molecular and physiological functions of Gβγ has significant implications.

331 citations

Journal ArticleDOI
20 Jul 2014-eLife
TL;DR: The dual requirement for Gβγ and PIP2 can help to explain why GIRK2 is activated by Gi/o, but not Gq coupled GPCRs.
Abstract: Though every cell in the body is surrounded by a membrane, there are a number of ways that molecules can pass through this membrane to either enter or leave the cell. Proteins from the GIRK family form channels in the membranes of mammalian cells, and when open these channels allow potassium ions to flow through the membrane to control the membrane's voltage. GIRK channels are found in the heart and in the central nervous system, and can be activated in a variety of ways. Sodium ions and molecules called ‘signaling lipids’ can regulate the activation of GIRK channels. These channels can also be caused to open by G proteins: proteins that are found inside cells and that help to transmit signals from the outside of a cell to the inside. Three G proteins—called Gα, Gβ, and Gγ—work together in a complex that functions a bit like a switch. When switched on, the Gα subunit is separated from the other two subunits (called Gβγ); and both parts can then activate different signaling pathways inside the cell. The Gβγ subunits and a signaling lipid have been known to regulate the opening of GIRK channels for a number of years, but these events have only been studied in the context of living cells. The specific role of each molecule, and whether the Gα subunit can also regulate the GIRK channels, remains unknown. Now Wang et al. have produced one type of mouse GIRK channel, called GIRK2, in yeast cells, purified this protein, and added it into an artificial membrane. This ‘reconstituted system’ allowed the regulation of a GIRK channel to be investigated under more controlled conditions than in previous experiments. Wang et al. found that the Gβγ subunits and the signaling lipid both need to be present to activate the GIRK2 channel. Sodium ions were not essential, but promoted further opening when Gβγ and the signaling lipid were already present. When locked in its ‘on’ state, the Gα subunit had no effect on GIRK2, but adding Gα locked in the ‘off’ state closed these channels by removing the Gβγ proteins. The findings of Wang et al. suggest that it should be possible to use a similar reconstituted system to investigate what allows different G proteins to activate specific signaling pathways.

327 citations

Journal ArticleDOI
12 Dec 1997-Science
TL;DR: The crystal structure of Gsalpha, the heterotrimeric G protein alpha subunit that stimulates adenylyl cyclase, was determined at 2.5 A in a complex with guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), suggesting that their effector specificity is primarily dictated by the shape of the binding surface formed by the switch II helix and the alpha3-beta5 loop.
Abstract: The crystal structure of Gsalpha, the heterotrimeric G protein alpha subunit that stimulates adenylyl cyclase, was determined at 2.5 A in a complex with guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). Gsalpha is the prototypic member of a family of GTP-binding proteins that regulate the activities of effectors in a hormone-dependent manner. Comparison of the structure of Gsalpha.GTPgammaS with that of Gialpha.GTPgammaS suggests that their effector specificity is primarily dictated by the shape of the binding surface formed by the switch II helix and the alpha3-beta5 loop, despite the high sequence homology of these elements. In contrast, sequence divergence explains the inability of regulators of G protein signaling to stimulate the GTPase activity of Gsalpha. The betagamma binding surface of Gsalpha is largely conserved in sequence and structure to that of Gialpha, whereas differences in the surface formed by the carboxyl-terminal helix and the alpha4-beta6 loop may mediate receptor specificity.

306 citations

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Performance
Metrics
No. of papers in the topic in previous years
YearPapers
20213
20205
20197
20187
20171
20168