Receptor-Mediated Activation of Gsa: Evidence for Intramolecular Signal Transduction

To investigate the mechanism by which cell surface receptors activate heterotrimeric G proteins, we applied a scanning mu-tagenesis approach to the carboxyl-terminal 40 % of as (resi-dues 236–394) to identify residues that play a role in receptor-mediated activation. We identified four regions of sequence in which mutations significantly impaired receptor-dependent stimulation of cAMP synthesis in transiently transfected cyc2 S49 lymphoma cells, which lack endogenous as. Residues at the carboxyl terminus are likely to be receptor contact sites. Buried residues near the bound GDP are connected to the carboxyl terminus by an a helix and may regulate GDP affinity. Residues in two adjacent loops of the GTPase domain at the interface with the helical domain, one of which includes a region, switch III, that changes conformation on GTP binding