G(I)3 DOES NOT CONTRIBUTE TO THE INHIBITION OF ADENYLATE-CYCLASE WHEN STIMULATION OF AN ALPHA-2-ADRENERGIC RECEPTOR CAUSES ACTIVATION OF BOTH G(I)2 AND G(I)3

Agonist occupancy of the alpha-2-C10 adrenergic receptor in a stable clone (1C) of Rat 1 fibroblasts produced by transfection of cells with genomic DNA encoding this receptor causes the activation of both of the pertussis-toxin-sensitive G-proteins G(i)2 and G(i)3 [Milligan, Carr, Gould, Mullaney & Lavan (1991) J. Biol. Chem. 266,6447-6455]. An IgG fraction from an antiserum (I3B) which identifies the C-terminal decapeptide of G(i)3-alpha only was able to inhibit partially receptor stimulation of high-affinity GTPase activity. An equivalent fraction from an antiserum (AS7) able to identify the C-terminal decapeptide of G(i)1-alpha + G(i)2-alpha, but not G(i)3-alpha, was also able to inhibit partially receptor stimulation of GTPase activity, and the effects of the two antisera were additive. By contrast, agonist-mediated inhibition of forskolin-amplified adenylate cyclase activity was abolished completely by the IgG fraction of antiserum AS7, but was not decreased by treatment with antiserum I3B. Based on the proportion of agonist-stimulated high-affinity GTPase which was prevented by each antiserum and on the measured membrane levels of G(i)2 and G(i)3, calculations indicated that essentially all of the cellular G(i)3, but only 15% of the available G(i)2, can be activated by the alpha-2-C10 adrenergic receptor in these cells. These results demonstrate that, although G(i)3 is activated by alpha-2-adrenergic agonists in membranes of clone 1C cells, it does not contribute to the transduction of receptor-mediated inhibition of adenylate cyclase.