Multiple regulatory roles of the carboxy terminus of Ste2p a yeast GPCR

Signaling and internalization of Ste2p, a model G protein-coupled receptor (GPCR) from the yeast Saccharomyces cerevisiae, are reported to be regulated by phosphorylation status of serine (S) and threonine (T) residues located in the cytoplasmic C-terminus. Although the functional roles of S/T residues located in certain C-terminus regions are relatively well characterized, systemic analyses have not been conducted for all the S/T residues that are spread throughout the C-terminus. A point mutation to alanine was introduced into the S/T residues located within three intracellular loops and the C-terminus individually or in combination. A series of functional assays such as internalization, FUS1-lacZ induction, and growth arrest were conducted in comparison between WT- and mutant Ste2p. The Ste2p in which all SIT residues in the C-terminus were mutated to alanine was more sensitive to alpha-factor, suggesting that phosphorylation in the C-terminus exerts negative regulatory activities on the Ste2p signaling. C-terminal SIT residues proximal to the seventh transmembrane domain were important for ligand-induced G protein coupling but not for receptor internalization. Sites on the central region of the C-terminus regulated both constitutive and ligand-induced internalization. Residues on the distal part were important for constitutive desensitization and modulated the G protein signaling mediated through the proximal part of the C-terminus. This study demonstrated that the C-terminus contains multiple functional domains with differential and interdependent roles in regulating Ste2p function in which the SIT residues located in each domain play critical roles. (C) 2011 Elsevier Ltd. All rights reserved.