α-Factor receptors from Saccharomyces cerevisiae are G-protein-coupled receptors containing seven transmembrane segments. The ability of α-factor receptors to form oligomeric complexes with each other and with other proteins was investigated. Both in vivo and in vitroevidence was obtained that suggests homo-oligomerization of receptors in the plasma membrane. When the membranes from cells coexpressing two differentially-tagged receptors were solubilized with detergent and subjected to immunoprecipitation, the antibodies specific for either epitope tag resulted in precipitation of both tagged species. Treatment of cultures with α-factor had little effect on the extent of oligomerization as judged by the sedimentation behavior of the receptor complexes and by the efficiency of coimmunoprecipitation. The ability of receptor complexes to undergo ligand-mediated endocytosis was evaluated by using membrane fractionation and fluorescence microscopy. Mutant receptors that fail to bind α-factor (Ste2-S184R) or lack the endocytosis signal (Ste2-T326) became competent for ligand-mediated endocytosis when they were expressed in cells containing wild-type receptors. Coimmunoprecipitation experiments indicated that the C-terminal cytoplasmic domain and intermolecular disulfide bonds were unnecessary for oligomer formation. Therefore, α-factor receptors form homo-oligomers and that these complexes are subject to ligand-mediated endocytosis. A crosslinking and immunoprecipitation strategy was used to capture and characterize the transient complexes that contain the α-factor receptor Ste2. Tagged receptors were crosslinked to form at least three high molecular weight complexes and the complexes were immunoprecipitated with antibodies against the tag. Western blotting analysis of the precipitated material revealed the presence of β and γ subunits of the heterotrimeric G protein, Ste4 and Stel8. Similar results were obtained when the cultures had been treated with α-factor prior to analysis. A truncated receptor missing most of the cytoplasmic C-terminal tail was also active in binding Ste4. Overall, these results constitute the first biochemical evidence for a physical association between the α-factor receptor and its cognate G-protein. Endocytic signals in the C-terminal tail (residues 297-431) of the α-factor receptor were analyzed. One signaling element, SINNDAKSS, (residues 331-339) is known to be sufficient (but not necessary) for endocytosis. Internal deletions of the STE2 gene were constructed that remove sequences encoding SINNDAKSS and selected regions of the C-terminal tail. Strains containing these alelles were then assayed for endocytosis in the presence and absence of α-factor. Residues from 360 to 431 were sufficient to mediate both constitutive and ligand-mediated endocytosis of the receptor even though 63 residues including the SINNDAKSS motif had been removed. Structural features of this region that were investigated further were the highly-ubiquitinated Lys374, the neighboring Lys387, and the GPFAD motif (residues 392-396). Lys374 and Lys387 were unnecessary for the element to promote exit from the plasma membrane; however, Lys374 may play some role in intracellular trafficking. The GPFAD motif was not sufficient to promote endocytosis, since the residues 360-399 provided no detectable endocytic activity. Overall, these results suggest that a new region in the C-terminal of the α-factor receptor, redundant with the SINNDAKSS motif, is sufficient to mediate the constitutive endocytosis as well as the ligand-mediated endocytosis of the receptor.

alpha-Factor receptors from Saccharomyces cerevisiae are G-protein-coupled receptors containing seven transmembrane segments. Receptors solubilized with the detergent n-dodecyl beta-D-maltoside were found to sediment as a single 8S species in glycerol density gradients. When the membranes from cells coexpressing two differentially tagged receptors were solubilized with detergent and subjected to immunoprecipitation, we found that the antibodies specific for either epitope tag resulted in precipitation of both tagged species. Coprecipitation was not a consequence of incomplete detergent extraction because the abundant plasma membrane protein Pma1 did not coprecipitate with the receptors. Moreover, the receptor complexes were present prior to detergent extraction because coimmunoprecipitation was not observed when cells expressing the single tagged species were mixed prior to membrane preparation. Treatment of cultures with alpha-factor had little effect on the extent of oligomerization as judged by the sedimentation behavior of the receptor complexes and by the efficiency of coimmunoprecipitation. The ability of receptor complexes to undergo ligand-mediated endocytosis was evaluated by using membrane fractionation and fluorescence microscopy. Mutant receptors that fail to bind alpha-factor (Ste2-S184R) or lack the endocytosis signal (Ste2-T326) became competent for ligand-mediated endocytosis when they were expressed in cells containing wild-type receptors. Coimmunoprecipitation experiments indicated that the C-terminal cytoplasmic domain and intermolecular disulfide bonds were unnecessary for oligomer formation. We conclude that alpha-factor receptors form homo-oligomers and that these complexes are subject to ligand-mediated endocytosis. Furthermore, we show for the first time that unoccupied receptors participate in these endocytosis-competent complexes.