Switch II Region in Gαi1: Specificity for Ric-8A

On the cell surface are G protein coupled receptors that bind to agonists, causing activation of intracellular G proteins, by catalyzing exchange of GTP for GDP at the G protein alpha subunit (Ga). G proteins are also activated by guanine nucleotide exchange factors (GEF) inside of the cell; these include Ric-8A and Ric-8B. GTP-bound Ga can stimulate the activity of intracellular enzymes. For example, Gas activates adenylyl cyclase, while Gai1 inhibits the activity of this enzyme. Biochemical studies have shown that Ric-8A is a GEF towards Gai1 whereas its isoform Ric-8B acts on Gas. Previous studies in our laboratory and others have shown that a region in Gαi1 called switch II binds to Ric-8A. In this study, we test the hypothesis that differences in amino acid sequence between Gai1 and Gas in switch II are responsible for the ability of these G proteins to discriminate between Ric-8A and Ric-8B. The switch regions of Gai1 and Gas differ in only three amino acids. We predict that, by mutating these amino acids in Gai1 to their corresponding residues in Gas, affinity for Ric-8A will be impaired. Single mutation primers (S206D, K209R and H213Q) were made, transformed and amplified through a polymerase chain reaction (PCR). Once mutant plasmid was expressed in E. coli cells, it is purified, and a tryptophan fluorescence assay is performed. This assay technique detects changes in the fluorescence of tryptophan 211, a side chain in switch II that is sensitive to the exchange of GTP for GDP. Our research sheds light on how mutants in Gai1 in the switch II region plays important role in specificity of binding for Ric-8A