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Molecular and Cellular Biology, July 2000, p. 5321-5329, Vol. 20, No. 14
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

The C Terminus of the Saccharomyces cerevisiae alpha -Factor Receptor Contributes to the Formation of Preactivation Complexes with Its Cognate G Protein

Mercedes Dosil,1,dagger Kimberly A. Schandel,2,Dagger Ekta Gupta,1 Duane D. Jenness,2 and James B. Konopka1,*

Department of Molecular Genetics and Microbiology, State University of New York, Stony Brook, New York 11794-5222,1 and Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, Massachusetts 016552

Received 28 February 2000/Returned for modification 13 April 2000/Accepted 24 April 2000

Binding of the alpha -factor pheromone to its G-protein-coupled receptor (encoded by STE2) activates the mating pathway in MATa yeast cells. To investigate whether specific interactions between the receptor and the G protein occur prior to ligand binding, we analyzed dominant-negative mutant receptors that compete with wild-type receptors for G proteins, and we analyzed the ability of receptors to suppress the constitutive signaling activity of mutant Galpha subunits in an alpha -factor-independent manner. Although the amino acid substitution L236H in the third intracellular loop of the receptor impairs G-protein activation, this substitution had no influence on the ability of the dominant-negative receptors to sequester G proteins or on the ability of receptors to suppress the GPA1-A345T mutant Galpha subunit. In contrast, removal of the cytoplasmic C-terminal domain of the receptor eliminated both of these activities even though the C-terminal domain is unnecessary for G-protein activation. Moreover, the alpha -factor-independent signaling activity of ste2-P258L mutant receptors was inhibited by the coexpression of wild-type receptors but not by coexpression of truncated receptors lacking the C-terminal domain. Deletion analysis suggested that the distal half of the C-terminal domain is critical for sequestration of G proteins. The C-terminal domain was also found to influence the affinity of the receptor for alpha -factor in cells lacking G proteins. These results suggest that the C-terminal cytoplasmic domain of the alpha -factor receptor, in addition to its role in receptor downregulation, promotes the formation of receptor-G-protein preactivation complexes.

* Corresponding author. Mailing address: Department of Molecular Genetics and Microbiology, State University of New York, Stony Brook, NY 11794-5222. Phone: (631) 632-8715. Fax: (631) 632-9797. E-mail: james.konopka{at}sunysb.edu.

dagger Present address: Departamento de Bioquímica y Biología Molecular, Universidad de Salamanca, Campus Unamuno, 37007 Salamanca, Spain.

Dagger Permanent address: Department of Natural Sciences, Assumption College, Worcester, MA 01615.

Molecular and Cellular Biology, July 2000, p. 5321-5329, Vol. 20, No. 14
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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