The mapping of yeast's G-protein coupled receptor with an atomic force microscope.

An atomic force microscope (AFM) can measure the adhesion force between a sample and a cantilever while simultaneously applying a rupture force during the imaging of a sample. An AFM should be useful in targeting specific proteins on a cell surface. The present study proposes the use of an AFM to measure the adhesion force between targeting receptors and their ligands, and to map the targeting receptors. In this study, Ste2p, one of the G protein-coupled receptors (GPCRs), was chosen as the target receptor. The specific force between Ste2p on a yeast cell surface and a cantilever modified with its ligand, α-factor, was measured and found to be approximately 250 pN. In addition, through continuous measuring of the cell surface, a mapping of the receptors on the cell surface could be performed, which indicated the differences in the Ste2p expression levels. Therefore, the proposed AFM system is accurate for cell diagnosis.

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.

Identification of specific transmembrane residues and ligand-induced interface changes involved in homo-dimer formation of a yeast G protein-coupled receptor.

The Saccharomyces cerevisiae alpha-factor pheromone receptor, Ste2p, has been studied as a model for G protein-coupled receptor (GPCR) structure and function. Dimerization has been demonstrated for many GPCRs, although the role(s) of dimerization in receptor function is disputed. Transmembrane domains one (TM1) and four (TM4) of Ste2p were shown previously to play a role in dimerization. In this study, single cysteine substitutions were introduced into a Cys-less Ste2p, and disulfide-mediated dimerization was assessed. Six residues in TM1 (L64 to M69) that had not been previously investigated and 19 residues in TM7 (T278 to A296) of which 15 were not previously investigated were mutated to create 25 single Cys-containing Ste2p molecules. Ste2p mutants V68C in TM1 and nine mutants in TM7 (cysteine substituted into residues 278, 285, 289, and 291 to 296) showed increased dimerization upon addition of an oxidizing agent in comparison to the background dimers formed by the Cys-less receptor. The formation of dimers was decreased for TM7 mutant receptors in the presence of alpha-factor indicating that ligand binding resulted in a conformational change that influenced dimerization. The effect of ligand on dimer formation suggests that dimers are formed in the resting state and the activated state of the receptor by different TM interactions.