INTERACTIONS OF THE ALPHA(2A)-ADRENOCEPTOR WITH MULTIPLE G(I)-FAMILY G-PROTEINS - STUDIES WITH PERTUSSIS TOXIN-RESISTANT G-PROTEIN MUTANTS

The alpha(2A)-adrenoceptor is the prototypic example of the family of G-protein-coupled receptors which function by activation of 'G(i)-like ' pertussis toxin-sensitive G-proteins. A number of members of this su bfamily of G-proteins are often co-expressed in a single cell type. To examine the interaction of this receptor with individual G(i)-family G-proteins the porcine alpha(2A)-adrenoceptor was transiently transfec ted into COS-7 cells either alone or with each of wild-type G(i)1 alph a, G(i)2 alpha and G(i)3 alpha or mutations of each of these G-protein s in which the cysteine residue which is the target for pertussis toxi n-catalysed ADP-ribosylation was exchanged for a glycine residue. The a,-adrenoceptor agonist UK14304 stimulated both high-affinity GTPase a ctivity and the binding of guanosine 5'-[gamma-(35)thio]-triphosphate (GTP[S-35]), when expressed without any additional G-protein. These ef fects were greatly reduced by pretreatment of the cells with pertussis toxin. Co-expression of each of the wild-type G(i)-like G-protein alp ha-subunits resulted in enhanced agonist activation of the cellular G- protein population which was fully prevented by pretreatment with pert ussis toxin. Co-expression of the receptor along with the cysteine-to- glycine mutations of G(i)1 alpha, G(i)2 alpha and G(i)3 alpha resulted in agonist stimulation of these G-proteins, which was as great as tha t of the wild type proteins, but now the agonist stimulation produced over that due to the activation of endogenously expressed G(i)-like G- proteins was resistant to pertussis toxin treatment. The Cys --> Gly m utations of G(i)1 alpha, G(i)2 alpha and G(i3)alpha were each also abl e to limit agonist-mediated stimulation of adenylate cyclase activity. The degree of agonist-mediated activation of the pertussis toxin-resi stant mutant of G(i)1a was correlated highly both with the level of ex pression of this G-protein and with the level of expression of the alp ha(2A)-adrenoceptor. Half-maximal stimulation of high-affinity GTPase activity of the Cys --> Gly mutants of G(i)1 alpha, G(i)2 alpha and G( i)3 alpha required 10-15-fold higher concentrations of agonist than di d stimulation of their wild-type counterparts, consistent with a model in which the affinity of functional interactions of the alpha(2A)-adr enoceptor with the wild-type G-protein is greater than with the pertus sis toxin-resistant mutant G-protein.