Showing papers on "GTP-Binding Protein alpha Subunits published in 1999"

Cyclic AMP-dependent protein kinase regulates pseudohyphal differentiation in Saccharomyces cerevisiae.

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.

Endomorphin-1 and endomorphin-2 show differences in their activation of mu opioid receptor-regulated G proteins in supraspinal antinociception in mice

Abstract: Endomorphin-1 and endomorphin-2 are tetrapeptides of the brain whose binding profiles and analgesic activities indicate that they are endogenous ligands at micro opioid receptors. To analyze the classes of G transducer proteins activated by these opioids in the production of supraspinal antinociception, the expression of alpha subunits of the G(i) protein class, G(i1), G(i2), G(i3), G(o1), G(o2), and G(z), and those of the G(q) protein family, G(q) and G(11), was reduced by administration of antisense oligodeoxynucleotides (ODNs) complementary to sequences in their respective mRNAs. The ODN treatments promoted differences in the analgesic effects displayed by morphine, [D-Ala(2),N-MePhe(4), Gly-ol(5)]enkephalin (DAMGO), and the novel opioids endomorphin-1 and endomorphin-2. The impairment of G(i1)alpha and G(i3)alpha function led to a weaker analgesic response to the endomorphins and to the alpha(2)-adrenoceptor agonist clonidine, whereas the effects of morphine and DAMGO were not affected. An antisense probe targeting G(i2)alpha blocked the antinociceptive effects of endomorphin-2, morphine, DAMGO, and clonidine but was without effect on the activity of endomorphin-1. Mice receiving the ODN to G(z)alpha subunits showed impaired response to all agonists. The knockdown of either G(o1)alpha, G(o2)alpha, G(q)alpha, or G(11)alpha had little or no influence on the antinociception induced by any of the opioids in the study. Thus, agonists exhibit differences in activating the variety of GTP-binding proteins regulated by mu opioid receptors.

The Yeast Trimeric Guanine Nucleotide-Binding Protein α Subunit, Gpa2p, Controls the Meiosis-Specific Kinase Ime2p Activity in Response to Nutrients

Abstract: Saccharomyces cerevisiae Gpa2p, the alpha subunit of a heterotrimeric guanine nucleotide-binding protein (G protein), is involved in the regulation of vegetative growth and pseudohyphal development. Here we report that Gpa2p also controls sporulation by interacting with the regulatory domain of Ime2p (Sme1p), a protein kinase essential for entrance of meiosis and sporulation. Protein-protein interactions between Gpa2p and Ime2p depend on the GTP-bound state of Gpa2p and correlate with down-regulation of Ime2p kinase activity in vitro. Overexpression of Ime2p inhibits pseudohyphal development and enables diploid cells to sporulate even in the presence of glucose or nitrogen. In contrast, overexpression of Gpa2p in cells simultaneously overproducing Ime2p results in a drastic reduction of sporulation efficiency, demonstrating an inhibitory effect of Gpa2p on Ime2p function. Furthermore, deletion of GPA2 accelerates sporulation on low-nitrogen medium. These observations are consistent with the following model. In glucose-containing medium, diploid cells do not sporulate because Ime2p is inactive or expressed at low levels. Upon starvation, expression of Gpa2p and Ime2p is induced but sporulation is prevented as long as nitrogen is present in the medium. The negative control of Ime2p kinase activity is exerted at least in part through the activated form of Gpa2p and is released as soon as nutrients are exhausted. This model attributes a switch function to Gpa2p in the meiosis-pseudohyphal growth decision.

Modulation of relative intrinsic activity of agonists at the alpha-2A adrenoceptor by mutation of residue 351 of G protein gi1alpha

Abstract: Compared with epinephrine, the relative intrinsic activity of a series of partial agonists to activate fusion proteins between the porcine alpha-2A adrenoceptor and the alpha-subunit of Gi1 was reduced after a single-point mutation (Cys351Gly) in the G protein. Although UK14304 was close to a full agonist at the fusion construct containing wild-type (Cys351)Gi1alpha, it was a partial agonist at that containing Gly351Gi1alpha. Moreover, although clonidine functioned as a good partial agonist to activate the fusion protein containing Cys351Gi1alpha, it was essentially an antagonist at the Gly351Gi1alpha-containing fusion protein. By contrast, incorporation of Ile351Gi1alpha into the fusion protein resulted in all partial agonists displaying higher intrinsic activity relative to epinephrine to activate this fusion protein than the one containing the wild-type G protein sequence. This is the first demonstration that the relative intrinsic activity of a series of agonists can be modified by a point mutation in a G protein rather than a receptor and indicates that the nature of a key contact site between a G protein and a receptor can selectively regulate partial agonist function. We provide a model for this based on the hydrophobicity of a key receptor-G protein alpha-subunit interaction interface.