1. The C terminus of the Saccharomyces cerevisiae alpha-factor receptor contributes to the formation of preactivation complexes with its cognate G protein.
- Author
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Dosil M, Schandel KA, Gupta E, Jenness DD, and Konopka JB
- Subjects
- Alleles, Amino Acid Substitution, Cytoplasm metabolism, GTP-Binding Protein alpha Subunits, Gq-G11, GTP-Binding Proteins genetics, Genes, Dominant, Genes, Lethal, Heterotrimeric GTP-Binding Proteins genetics, Heterotrimeric GTP-Binding Proteins metabolism, Ligands, Mutation, Receptors, Mating Factor, Receptors, Peptide genetics, Saccharomyces cerevisiae genetics, Signal Transduction, GTP-Binding Protein alpha Subunits, GTP-Binding Proteins metabolism, Receptors, Peptide metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins, Transcription Factors
- Abstract
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.
- Published
- 2000
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