Differential interactions of fluorescent agonists and antagonists with the yeast G protein coupled receptor Ste2p.

We describe a rapid method to probe for mutations in cell surface ligand-binding proteins that affect the environment of bound ligand. The method uses fluorescence-activated cell sorting to screen randomly mutated receptors for substitutions that alter the fluorescence emission spectrum of environmentally sensitive fluorescent ligands. When applied to the yeast α-factor receptor Ste2p, a G protein-coupled receptor, the procedure identified 22 substitutions that red shift the emission of a fluorescent agonist, including substitutions at residues previously implicated in ligand binding and at additional sites. A separate set of substitutions, identified in a screen for mutations that alter the emission of a fluorescent α-factor antagonist, occurs at sites that are unlikely to contact the ligand directly. Instead, these mutations alter receptor conformation to increase ligand-binding affinity and provide signaling in response to antagonists of normal receptors. These results suggest that receptor--agonist interactions involve at least two sites, of which only one is specific for the activated conformation of the receptor.

In vitro characterization of ligand-induced oligomerization of the S. cerevisiae G-protein coupled receptor, Ste2p.

BACKGROUND: The S. cerevisiae alpha-factor receptor, Ste2p, is a G-protein coupled receptor that plays key roles in yeast signaling and mating. Oligomerization of Ste2p has previously been shown to be important for intracellular trafficking, receptor processing and endocytosis. However the role of ligand in receptor oligomerization remains enigmatic. METHODS: Using functional recombinant forms of purified Ste2p, atomic force microscopy, dynamic light scattering and chemical crosslinking are applied to investigate the role of ligand in Ste2p oligomerization. RESULTS: Atomic force microscopy images indicate a molecular height for recombinant Ste2p in the presence of alpha-factor nearly double that of Ste2p alone. This observation is supported by complementary dynamic light scattering measurements which indicate a ligand-induced increase in the polydispersity of the Ste2p hydrodynamic radius. Finally, chemical cross-linking of HEK293 plasma membranes presenting recombinant Ste2p indicates alpha-factor induced stabilization of the dimeric form and higher order oligomeric forms of the receptor upon SDS-PAGE analysis. CONCLUSIONS: alpha-factor induces oligomerization of Ste2p in vitro and in membrane. GENERAL SIGNIFICANCE: These results provide additional evidence of a possible role for ligand in mediation of Ste2p oligomerization in vivo.