The yeast Gα subunit Gpa2p and its coupled receptor Gpr1p function in a signaling pathway that is required for the transition to pseudohyphal and invasive growth. A two-hybrid screen using a constitutively active allele of GPA2 identified the KRH1 gene as encoding a potential binding partner of Gpa2p. Strains containing deletions of KRH1 and its homolog KRH2 were hyper-invasive and displayed a high level of expression of FLO11, a gene involved in pseudohyphal and invasive growth. Therefore, KRH1 and KRH2 encode negative regulators of the invasive growth pathway. Cells containing krh1Δ krh2Δ mutations also displayed increased sensitivity to heat shock and decreased sporulation efficiency, indicating that Krh1p and Krh2p regulate multiple processes controlled by the cAMP/PKA pathway. The krh1Δ krh2Δ mutations suppressed the effect of a gpa2Δ mutation on FLO11 expression and eliminated the effect of a constitutively active GPA2 allele on induction of FLO11 and heat shock sensitivity, suggesting that Krh1p and Krh2p act downstream of Gpa2p. The Sch9p kinase was not required for the signal generated by deletion of KRH1 and KRH2; however, the cAMP-dependent kinase Tpk2p was required for generation of this signal. These results support a model in which activation of Gpa2p relieves the inhibition exerted by Krh1p and Krh2p on components of the cAMP/PKA signaling pathway.

Pseudohyphal and invasive growth in the yeast Saccharomyces cerevisiae is regulated by the kelch repeatcontaining proteins Gpb1p and Gpb2p, which act downstream of the G protein ␣-subunit Gpa2p. Here we show that deletion of GPB1 and GPB2 causes increased haploid invasive growth in cells containing any one of the three protein kinase A (PKA) catalytic subunits, suggesting that Gpb1p and Gpb2p are able to inhibit each of these kinases. Cells containing gpb1⌬ gpb2⌬ mutations also display increased phosphorylation of the PKA substrates Sfl1p and Msn2p, indicating that Gpb1p and Gpb2p are negative regulators of PKA substrate phosphorylation. Stimulation of PKA-dependent signaling by gpb1⌬ gpb2⌬ mutations occurs in cells that lack both adenylyl cyclase and the high-affinity cyclic AMP (cAMP) phosphodiesterase. This effect is also seen in cells that lack the low-affinity cAMP phosphodiesterase. Given that these three enzymes control the synthesis and degradation of cAMP, these results indicate that the effect of Gpb1p and Gpb2p on PKA substrate phosphorylation does not occur by regulating the intracellular cAMP concentration. These findings suggest that Gpb1p and Gpb2p mediate their effects on the cAMP/PKA signaling pathway either by inhibiting the activity of PKA in a cAMP-independent manner or by activating phosphatases that act on PKA substrates.

Kelch repeat proteins Gpb1 and Gpb2 control yeast PKA activity in response to nutrients by stimulating phosphorylation of the Bcy1 regulatory subunit. Gpb1 and Gpb2 function by blocking inhibition of Bcy1 phosphorylation by PKA catalytic subunits. Phosphorylated Bcy1 is more stable and is a more effective inhibitor of PKA activity.

Rex1(Zfp42), GeneID 132625, is a gene whose expression is closely associated with pluripotency/multipotency in both mouse and human embryonic stem cells. To study the function of the murine Rex1 gene in vivo, we have used cre/lox technology to create Rex1(floxed) mice and mice deficient in Rex1 gene function. Rex1-/- males are characterized by an age-associated decrease in sperm counts, abnormal sperm morphology, and mild testicular atrophy. We characterized global patterns of gene expression in primary germ cells by microarray and identified the growth hormone responsive gene, GRTP1, as a transcript present at a 4.5 fold higher level in wild type (WT) compared to Rex1-/- mice. We analyzed immature germ cell (Dazl), proliferating (PCNA), and Sertoli cell populations, and quantitated levels of apoptosis in Rex1-/- as compared to WT testes. We evaluated the expression of proteins previously reported to correlate with Rex1 expression, such as STAT3, phospho-STAT3, p38, and phospho-p38 in the testis. We report a distinct cellular localization of total STAT3 protein in Rex1-/- affected testes. Our data suggest that loss of Rex1 leads to impaired testicular function.

The kelch repeat-containing proteins Krh1p and Krh2p are negative regulators of the Gpa2p signaling pathway that directly interact with the G protein ␣-subunit Gpa2p in the yeast Saccharomyces cerevisiae. A screen was carried out to identify Gpa2p variants that are defective in their ability to bind Krh1p but retain the ability to bind another Gpa2p-interacting protein, Ime2p. This screen identified amino acids Gln-419 and Asn-425 as being important for the interaction between Gpa2p and Krh1p. Gpa2p variants with changes at these positions are defective for Krh1p binding in vivo. Cells containing these forms of Gpa2p display decreased heat shock resistance and increased expression of a gene required for pseudohyphal growth. These findings indicate that the substitutions at positions 419 and 425 confer a degree of constitutive activity to the Gpa2p ␣-subunit. Residues Gln-419 and Asn-425 are located in the ␤6-␣5 loop and ␣5 helix of Gpa2p, which is the region that couples receptor binding to guanine nucleotide exchange. The results suggest that binding of Gpa2p to Krh1p does not resemble the binding of G␣ subunits to either G␤ subunits or effectors, but it instead represents a novel type of functional interaction.