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Eukaryotic Cell, September 2008, p. 1540-1548, Vol. 7, No. 9
1535-9778/08/$08.00+0     doi:10.1128/EC.00154-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

The RGS Protein Crg2 Regulates Pheromone and Cyclic AMP Signaling in Cryptococcus neoformans{triangledown} ,{dagger}

Gui Shen,1 Yan-Li Wang,1 Amy Whittington,2 Lie Li,1,{ddagger} and Ping Wang1,2,3*

The Research Institute for Children,1 Departments of Microbiology, Immunology, and Parasitology,2 Pediatrics, Louisiana State University Health Sciences Center, New Orleans, Louisiana 701183

Received 2 May 2008/ Accepted 15 July 2008

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{alpha} subunits Gpa2 and Gpa3. It has also been proposed that Crg2 has a role in mating, as genetic crosses involving {Delta}crg2 mutants resulted in formation of hyperfilaments. We found that mutation of Gpa2 and Gpa3 partially suppressed the hyperfilamentation, mutation of Gpa3 alleviated {Delta}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 {Delta}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.

* Corresponding author. Mailing address: Children's Hospital, Research & Education Bldg., Rm. 3123, New Orleans, LA 70118. Phone: (504) 896-2739. Fax: (504) 894-5379. E-mail: pwang{at}lsuhsc.edu

{triangledown} Published ahead of print on 25 July 2008.

{dagger} Supplemental material for this article may be found at http://ec.asm.org/.

{ddagger} Present address: University of Illinois at Chicago, 835 S. Wolcott Ave., Chicago, IL 60612.

Eukaryotic Cell, September 2008, p. 1540-1548, Vol. 7, No. 9
1535-9778/08/$08.00+0     doi:10.1128/EC.00154-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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