The cAMP-PKA pathway consists of an extracellular ligand-sensitive G protein-coupled receptor, a G protein signal transmitter, and the effector, adenylate cyclase, of which the product, cAMP, acts as an intracellular second messenger. cAMP activates PKA by dissociating the regulatory subunit from the catalytic subunit. Yeast cells (Saccharomyces cerevisiae) contain a glucose͞sucrose-sensitive seven-transmembrane domain receptor, Gpr1, that was proposed to activate adenylate cyclase through the G␣ protein Gpa2. Consistently, we show here that adenylate cyclase binds only to active, GTP-bound Gpa2. Two related kelch-repeat proteins, Krh1͞ Gpb2 and Krh2͞Gpb1, are associated with Gpa2 and were suggested to act as G␤ mimics for Gpa2, based on their predicted seven-bladed ␤-propeller structure. However, we find that although Krh1 associates with both GDP and GTP-bound Gpa2, it displays a preference for GTP-Gpa2. The strong down-regulation of PKA targets by Krh1 and Krh2 does not require Gpa2 but is strictly dependent on both the catalytic and the regulatory subunits of PKA. Krh1 directly interacts with PKA by means of the catalytic subunits, and Krh1͞2 stimulate the association between the catalytic and regulatory subunits in vivo. Indeed, both a constitutively active GPA2 allele and deletion of KRH1͞2 lower the cAMP requirement of PKA for growth. We propose that active Gpa2 relieves the inhibition imposed by the kelch-repeat proteins on PKA, thereby bypassing adenylate cyclase for direct regulation of PKA. Importantly, we show that Krh1͞2 also enhance the association between mouse R and C subunits, suggesting that Krh control of PKA has been evolutionarily conserved.Saccharomyces cerevisiae ͉ signal transduction ͉ glucose

The cAMP-protein kinase A (PKA) pathway in the yeast Saccharomyces cerevisiae controls a variety of properties that depend on the nutrient composition of the medium. High activity of the pathway occurs in the presence of rapidly fermented sugars like glucose or sucrose, but only as long as growth is maintained. Growth arrest of fermenting cells or growth on a respiratory carbon source, like glycerol or ethanol, is associated with low activity of the PKA pathway. We have studied how different nutrients trigger rapid activation of the pathway. Glucose and sucrose activate cAMP synthesis through a G-protein-coupled receptor system, consisting of the GPCR Gpr1, the Galpha protein Gpa2 and its RGS protein Rgs2. Glucose is also sensed intracellularly through its phosphorylation. Specific mutations in Gpr1 abolish glucose but not sucrose signalling. Activation of the PKA pathway by addition of a nitrogen source or phosphate to nitrogen- or phosphate-starved cells, respectively, is not mediated by an increase in cAMP. Activation by amino acids is triggered by the general amino acid permease Gap1, which functions as a transporter/receptor. Short truncation of the C-terminus results in constitutively activating alleles. Activation by ammonium uses the ammonium permeases Mep1 and Mep2 as receptor. Specific point mutations in Mep2 uncouple signalling from transport. Activation by phosphate is triggered a.o. by the Pho84 phosphate permease. Several mutations in Pho84 separating transport and signalling or triggering constitutive activation have been obtained.

Recent microarray analysis has revealed multiple levels of genomic sensitivity to glucose and highlighted the power of genome-wide analysis to detect cellular responses to minute environmental changes in the yeast Saccharomyces cerevisiae.