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Eukaryotic Cell doi:10.1128/EC.00154-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

The RGS Protein Crg2 Regulates Pheromone and cAMP Signaling in Cryptococcus neoformans

The Research Institute for Children, Departments of Microbiology, Immunology, and Parasitology, and Pediatrics, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70118 USA

^* To whom correspondence should be addressed. Email: pwang{at}lsuhsc.edu.

	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. Crg2 is proposed to also have 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 MAP 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 form enlarged capsules that mimic cells expressing a constitutively active GPA1^Q284L allele, and that the levels of intracellular cAMP are also elevated, suggesting that Crg2 also negatively regulates the Gpa1-cAMP signaling pathway. We further showed that Crg2 interacts with Gpa3 and Gpa1, but not Gpa2 in a pull down assay, and Crg2 maintains a higher in vitro GAP 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 protein of multi-regulatory function, including one that controls mating distinctly from Crg1, and one that serves as a novel inhibitor of Gpa1-cAMP signaling.