No abstract

Functional analysis of polycystin-1, the product of the gene most frequently mutated in autosomal dominant polycystic kidney disease, has revealed that this protein is involved in the regulation of diverse signaling pathways such as the activation of the transcription factor AP-1 and modulation of Wnt signaling. However, the initial steps involved in the activation of such cascades have remained unclear. We demonstrated previously that the C-terminal cytosolic tail of polycystin-1 binds and activates heterotrimeric G proteins in vitro. To test if polycystin-1 can activate cellular signaling cascades via heterotrimeric G protein subunits, polycystin-1 Cterminal tail-mediated c-Jun N-terminal kinase (JNK) and AP-1 activities were assayed in transiently transfected 293T cells in the presence of dominant-negative, G protein inhibiting constructs, and in the presence of cotransfected G␣ subunits. The results showed that polycystin-1-mediated JNK/AP-1 activation is mediated by G␣ and G␤␥ subunits. Polycystin-1-mediated AP-1 activity could be significantly augmented by cotransfected G␣ i , G␣ q , and G␣ 12/13 subunits, suggesting that polycystin-1 can couple with and activate several heterotrimeric G protein families.

According to the prevailing paradigm, G-proteins are composed of three subunits, an ␣ subunit with GTPase activity and a tightly associated ␤␥ subunit complex. In the yeast Saccharomyces cerevisiae there are two known G␣ proteins (Gpa1 and Gpa2) but only one G␤␥, which binds only to Gpa1. Here we show that the yeast ortholog of RACK1 (receptor for activated protein kinase C1) Asc1 functions as the G␤ for Gpa2. As with other known G␤ proteins, Asc1 has a 7-WD domain structure, interacts directly with the G␣ in a guanine nucleotide-dependent manner, and inhibits G␣ guanine nucleotide exchange activity. In addition, Asc1 binds to the effector enzyme adenylyl cyclase (Cyr1), and diminishes the production of cAMP in response to glucose stimulation. Thus, whereas Gpa2 promotes glucose signaling through elevated production of cAMP, Asc1 has opposing effects on these same processes. Our findings reveal the existence of an unusual G␤ subunit, one having multiple functions within the cell in addition to serving as a signal transducer for cell surface receptors and intracellular effectors.

These findings reveal a novel phosphorylation-dependent feedback loop leading to diminished cellular responses to an osmotic-stress stimulus.

A common property of G protein-coupled receptors is that they become less responsive with prolonged stimulation. Regulators of G protein signaling (RGS proteins) are well known to accelerate G protein GTPase activity and do so by stabilizing the transition state conformation of the G protein ␣ subunit. In the yeast Saccharomyces cerevisiae there are four RGS-homologous proteins (Sst2, Rgs2, Rax1, and Mdm1) and two G␣ proteins (Gpa1 and Gpa2). We show that Sst2 is the only RGS protein that binds selectively to the transition state conformation of Gpa1. The other RGS proteins also bind Gpa1 and modulate pheromone signaling, but to a lesser extent and in a manner clearly distinct from Sst2. To identify other candidate pathway regulators, we compared pheromone responses in 4,349 gene deletion mutants representing nearly all nonessential genes in yeast. A number of mutants produced an increase (sst2, bar1, asc1, and ygl024w) or decrease (cla4) in pheromone sensitivity or resulted in pheromone-independent signaling (sst2, pbs2, gas1, and ygl024w). These findings suggest that Sst2 is the principal regulator of Gpa1-mediated signaling in vivo but that other proteins also contribute in distinct ways to pathway regulation.G protein-coupled receptors respond to a vast array of chemical and sensory signals, including hormones, neurotransmitters, odors, and light. Approximately one-third of all drugs act by binding directly to receptors of this class (64). Upon agonist stimulation of the receptor, a cognate G protein ␣ subunit will exchange GDP for GTP and undergo dissociation from the G protein ␤␥ subunit dimer. The dissociated subunits bind to effector enzymes, which in turn activate protein kinases, trigger new gene transcription, and ultimately produce programmed changes in cell homeostasis or differentiation (90). Regulators of G protein signaling (RGS proteins) function as GTPase-accelerating proteins (GAPs) and, in this manner, promote rapid inactivation or desensitization of the signal (89).Whereas mammalian genome analysis has revealed at least 16 G␣-and ϳ40 RGS-encoding genes (89, 106), a similar analysis in the yeast Saccharomyces cerevisiae reveals only two G␣ subunits but at least four RGS protein homologues. Gpa1 mediates cellular responses to mating pheromones. These pheromones, called a-factor and ␣-factor, are produced by haploid a and ␣ cells and bind to G protein-coupled receptors on cells of the opposite mating type. Upon activation of pheromone receptors, Gpa1 binds to GTP and dissociates from the G␤␥ dimer Ste4/Ste18, and the dissociated subunits activate a multitude of downstream effectors leading to cell fusion (mating) to form an a/␣ diploid (36, 50). Prominent among the known effectors are components of a MAP (mitogen-activated protein) kinase cascade comprised of Ste20, Ste11, Ste7, and Fus3. A parallel signaling pathway responds to glucose stimulation, leading to activation of a distinct receptor (Gpr1) (66,73,76,99,124), a distinct G protein ␣ subunit (Gpa2), and an atypical G protein ␤␥ complex ...