Accessibility of cysteine residues substituted into the cytoplasmic regions of the alpha-factor receptor identifies the intracellular residues that are available for G protein interaction.

The S. cerevisiae α-factor pheromone receptor (Ste2) belongs to the large family of Gprotein-coupled receptors (GPCRs) that transduce the signals for light, taste, olfaction, and many biomedically important hormones. Although GPCRs are quite diverse in sequence, they function in a similar manner to activate the α subunit of heterotrimeric G proteins to bind GTP and they also show similar structural architecture in that they are composed of a bundle of seven transmembrane domains (TMDs) connected by extracellular loops and intracellular loops (1, 2). Ste2 shows overall similarity to many mammalian GPCRs in that the core region containing the TMDs is involved in signal transduction and the C terminal tail is a target for post-translational modifications that regulate receptor desensitization and endocytosis (3). Ste2 activates a heterotrimeric G protein in which the α subunit shows about 45% identity with mammalian Gα proteins and is most closely related to the Gi subfamily (4). In addition, comparison of Ste2 with rhodopsin, a member of the large class A subfamily of mammalian GPCRs, indicates that there are similar microdomains in these divergent receptors (5). These results suggest that there are underlying similarities in the mechanisms of signal transduction by the diverse GPCR family. A major area of interest is to determine how the intracellular regions of GPCRs interact with G proteins to promote GTP exchange on Gα. As part of this interaction, the intracellular loops and ends of the TMDs are thought to form a pocket that interacts with the C terminus of Gα to promote GTP exchange (1, 6). Mutagenesis studies indicate that Ste2 is similar to other GPCRs in that the third intracellular loop plays an important role in G protein activation (7-10). However, Alanine scanning and random mutagenesis of the third loop region found that relatively conservative mutations did not result in strong defects, making it difficult to implicate specific residues as being functionally important (9, 10). The Ste2 3rd loop mutations that most strongly affect signaling typically involve substitutions that result in a gain or loss of polar side chains (7-9). Similar complexity has been observed for mammalian GPCRs. Mutagenesis studies have implicated certain regions, such as the N and C termini of the third loop that border TMDs 5 and 6, but the function of these residues is often not conserved even in closely related GPCRs (1, 11). To better define the Ste2 residues capable of interacting with G protein, in this study we examined the solvent accessibility of residues on the intracellular side of the receptor. Cys substitution mutations were introduced into the residues that span across the intracellular loops and the cytoplasmic end of TMD7 so that their solvent accessibility could be probed with MTSEA-biotin, a thiol reactive probe that does not react with membrane-buried residues (12). A similar approach used to define that topology of the extracellular regions of Ste2 demonstrated that the extracellular ends of the TMDs play a key role in ligand binding and in promoting the active receptor conformation (13, 14), and also provided a structural framework for comparing Ste2 to mammalian GPCRs (5). Therefore, the accessibility of residues in the cytoplasmic regions of Ste2 was assayed to provide a structural framework for understanding the role of residues that function in G protein activation.