Mechanisms Governing the Activation and Trafficking of Yeast G Protein-coupled Receptors

Abstract

We have addressed the mechanisms governing the activation and trafficking of G protein-coupled receptors (GPCRs) by analyzing constitutively active mating pheromone receptors (Ste2p and Ste3p) of the yeast Saccharomyces cerevisiae. Substitution of the highly conserved proline residue in transmembrane segment VI of these receptors causes constitutive signaling. This proline residue may facilitate folding of GPCRs into native, inactive conformations, and/or mediate agonist-induced structural changes leading to G protein activation. Constitutive signaling by mutant receptors is suppressed upon coexpression with wild-type, but not G protein coupling-defective, receptors. Wild-type receptors may therefore sequester a limiting pool of G proteins; this apparent "precoupling" of receptors and G proteins could facilitate signal production at sites where cell surface projections form during mating partner discrimination. Finally, rather than being expressed mainly at the cell surface, constitutively active pheromone receptors accumulate in post-endoplasmic reticulum compartments. This is in contrast to other defective membrane proteins, which apparently are targeted by default to the vacuole. We suggest that the quality-control mechanism that retains receptors in post-endoplasmic reticulum compartments may normally allow wild-type receptors to fold into their native, fully inactive conformations before reaching the cell surface. This may ensure that receptors do not trigger a response in the absence of agonist.

Oligomerization, Biogenesis, and Signaling Is Promoted by a Glycophorin A-like Dimerization Motif in Transmembrane Domain 1 of a Yeast G Protein-coupled Receptor

“…Homophilic interaction between receptor point mutants was studied by co-expression of receptors from their native STE2 promoter on the high copy plasmids pRS423STE2⌬tail-YFP and pRS424STE2⌬tail-CFP (29). Receptor mutants used for assays of agonist signaling and binding retained the full-length C-terminal tail and were expressed from the native STE2 promoter on a single copy plasmid pRS314STE2-3xmyc (30). All mutations were created by use of the Stratagene QuikChange TM site-directed mutagenesis kit.…”

Subunits of a Yeast Oligomeric G Protein-coupled Receptor Are Activated Independently by Agonist but Function in Concert to Activate G Protein Heterotrimers

“…Cells were grown in synthetic medium containing adenine and amino acid supplements, but lacking tryptophan, histidine, or uracil to maintain plasmids. All mutations were made in the single-copy plasmid pRS314Ste2-3myc (39), which uses native STE2 promoter and terminator sequences to drive expression of full-length or C-terminally truncated (at codon 303) ␣-factor receptors tagged at their C termini with three copies of the c-Myc epitope.…”

“…Preparation of 35 S-labeled ␣-Factor and Agonist-binding AssaysMethods used to purify 35 S-labeled ␣-factor from metabolically labeled yeast cells and to perform ligand-binding assays with inviable, intact cells are identical to those described previously (39,41). Agonist binding data were analyzed according to the method of Scatchard and fitted by nonlinear least-squares regression.…”

Dual Lipid Modification Motifs in Gαand GγSubunits Are Required for Full Activity of the Pheromone Response Pathway inSaccharomyces cerevisiae

“…The STE2 disruptions were created in CMY23 and CMY24 using the integrating plasmid YIpste2), which contains the 5Ј-and 3Ј-untranslated regions of STE2 but no coding sequence (Stefan et al, 1998). The plasmid was digested with ClaI and transformed into yeast.…”

“…The SST1 gene was disrupted using plasmid pJGsst1) (Reneke et al, 1988) digested with SalI and EcoRI and transformed into yeast strains to create CMY23 and CMY24. Successful disruptions (as assayed by halo assays) were streaked on media containing 5Ј-fluoro-orotic acid, so that the STE2 disruption could be performed.The STE2 disruptions were created in CMY23 and CMY24 using the integrating plasmid YIpste2), which contains the 5Ј-and 3Ј-untranslated regions of STE2 but no coding sequence (Stefan et al, 1998). The plasmid was digested with ClaI and transformed into yeast.…”