The RGS Protein Crg2 Regulates Pheromone and Cyclic AMP Signaling in Cryptococcus neoformans

Abstract

Crg1 and Crg2 are regulators of G-protein signaling homologs found in the human fungal pathogen Cryptococcus neoformans. Crg1 negatively regulates pheromone responses and mating through direct inhibition of G␣ subunits Gpa2 and Gpa3. It has also been proposed that Crg2 has a role in mating, as genetic crosses involving ⌬crg2 mutants resulted in formation of hyperfilaments. We found that mutation of Gpa2 and Gpa3 partially suppressed the hyperfilamentation, mutation of Gpa3 alleviated ⌬crg2-specfic cell swelling, and mutation of the mitogen-activated protein kinase Cpk1 blocked both processes. These findings indicate that Gpa2 and Gpa3 function downstream of Crg2 and that Gpa3 is also epistatic to Crg2 in a Cpk1-dependent morphogenesis process linked to mating. Significantly, we found that ⌬crg2 mutants formed enlarged capsules that mimic cells expressing a constitutively active GPA1(Q284L) allele and that the levels of intracellular cyclic AMP (cAMP) were also elevated, suggesting that Crg2 also negatively regulates the Gpa1-cAMP signaling pathway. We further showed that Crg2 interacted with Gpa3 and Gpa1, but not Gpa2, in a pulldown assay and that Crg2 maintained a higher in vitro GTPase-activating protein activity toward Gpa3 and Gpa1 than to Gpa2. Finally, we found that dysregulation of cAMP due to the Crg2 mutation attenuated virulence in a murine model of cryptococcosis. Taken together, our study reveals Crg2 as an RGS (regulator of G-protein signaling) protein of multiregulatory function, including one that controls mating distinctly from Crg1 and one that serves as a novel inhibitor of Gpa1-cAMP signaling.Regulators of G-protein signaling (RGS) are a family of proteins that share the highly conserved RGS domain of about 125 amino acid residues and play an important role in cell communication among eukaryotic organisms. The RGS domain allows it to interact specifically with the cognate G␣ to promote signaling inactivation through acceleration of G␣-bound GTP to GDP hydrolysis (7,8,10,15,21,34). In humans, more than 20 RGS proteins have been reported, which are also known to have at least 20 G␣ subunits, 6 G␤ subunits, and 11 G␥ subunits (20,31,43). In the lower eukaryotes, such as the budding yeast Saccharomyces cerevisiae that contains two G␣ subunits, one G␤ subunit, and one G␥ subunit, four RGS and RGS-like proteins, including Sst2, Rgs2, Rax1, and Mdm1, have been described. Sst2 is an archetypical RGS protein that functions as a GTPase-activating protein (GAP) to inhibit G␣ Gpa1 and promote pheromone desensitization (3, 9), whereas Rgs2, identified through a screen for multicopy suppressors of Gpa2-mediated glucose-induced loss of heat resistance, was shown to act as a negative regulator of Gpa2 and glucoseinduced cyclic AMP (cAMP) signaling (38). Although Rax1 (return to axial) and Mdm1, a multidomain-containing protein similar to mammalian sorting nexin 13, contain the conserved RGS and RGS-like domains, there is no evidence demonstrating their direct involvement in G-protein signaling (13,27,28).The...

A Ric8/Synembryn Homolog Promotes Gpa1 and Gpa2 Activation To Respectively Regulate Cyclic AMP and Pheromone Signaling in Cryptococcus neoformans

“…While we were unable to detect an interaction between Gpa3 and Ste3␣, Hsueh et al were able to establish an interaction using a different version of Y2H screening (13). Finally, Gpa3 is able to interact with Crg2, which is the main negative regulator of Gpa1 in cAMP signaling (31,32).…”

“…Several RGS proteins, including Crg1 and Crg2, have also been described (31,32). While Gpb1 couples only with Gpa2, no conventional G␤ protein that couples with Gpa1 has been found.…”

Regulatory Circuitry Governing Fungal Development, Drug Resistance, and Disease

“…Crg2 also negatively regulates both Gpa2 and Gpa3, although it is also capable of acting as a GAP of Gpa1, downregulating the cAMP-PKA signaling cascade (529). Consequently, crg2 mutants exhibit defects in filamentation and are highly attenuated in virulence (529,626).…”

The Cryptococcus neoformans Capsule: a Sword and a Shield

“…The pathway also has a number of negative-feedback elements, including the Crg2 regulator of G-protein signaling, which interacts directly with Gpa1 to limit cAMP production (180,219). Additionally, the Pde1 phosphodiesterase negatively regulates the pathway by degrading intracellular cAMP (90).…”