Measurement of Agonist-Dependent and-Independent Signal Initiation of 1b-Adrenoceptor Mutants by Direct Analysis of Guanine Nucleotide Exchange on the G Protein G11

Immunoprecipitation of a fusion protein between the 1b-adre-noceptor and G11 following a [ 35S]GTPS [guanosine-5-O-(3-thio)triphosphate] binding assay resulted in incorporation of low levels of nucleotide. The agonist phenylephrine increased in-corporation some 30-fold. Agonist-induced binding repre-sented 1.0 mol of [35S]GTPS/mol of fusion protein. This was to the G protein linked to the receptor rather than endogenous Gq/G11 as a fusion protein containing the 1b-adrenoceptor and a form of G11 (G 208A) unable to exchange guanine nucle-otides effectively, bound [35S]GTPS very poorly. Fusion pro-teins between A293E, D142A, and 3CAM mutants of the 1b-adrenoceptor and G11 bound substantially greater levels o