IMMUNOPRECIPITATION OF ALPHA-2A-ADRENERGIC RECEPTOR-GTP-BINDING PROTEIN COMPLEXES USING GTP-BINDING PROTEIN SELECTIVE ANTISERA - CHANGES IN RECEPTOR/GTP-BINDING PROTEIN-INTERACTION FOLLOWING AGONIST BINDING

The nature of the interaction of cloned alpha-2a-adrenergic receptors from LLC-PK1-O clone cells with G proteins was investigated using an immunoprecipitation approach. Following solubilization of the alpha-2a receptors, antiserum 8730, which is directed against the C-terminal region of G(i-alpha), immunoprecipitated alpha-2a receptor-G(i)alpha complexes. The immunoprecipitation was specific since it could be blocked by the peptide to which antiserum 8730 was generated. Antisera 3646 (anti-G(i-alpha-1), 1521 (anti-G(i-alpha-2) and 1518 (anti-G(i-alpha-3) immunoprecipitated solubilized alpha-2a receptor-G(i-alpha) complexes, indicating that all three G(i-alpha) subtypes couple with the alpha-2a receptor. Antiserum 9072, which is directed against the C-terminal region of G(o-alpha), immunoprecipitated solubilized alpha-2a receptor-G(alpha) complexes indicating that these receptors are also coupled to G(o-alpha). Antiserum 8132, which is directed against G(beta-36), immunoprecipitated solubilized alpha-2a receptors while the G(beta-35) antiserum 8129, did not, indicating that alpha-2a receptors selectively associate with G(beta-36). The binding of the partial agonist p-aminoclonidine to the solubilized alpha-2a receptor alters the association of the receptor with G proteins. Following p-aminoclonidine binding to the solubilized alpha-2a receptor, the ability of the C-terminal directed G(alpha) antisera 8730 and 9072 to coimmunoprecipitate the alpha-2a receptor-G(alpha) complex was greatly reduced. The effect of p-aminoclonidine was concentration dependent, mimicked by the full agonist UK 14304 and blocked by the alpha-2 receptor antagonist yohimbine. In contrast, antisera directed against internal regions of G(i-alpha) and G(o-alpha), immunoprecipitated the agonist-bound and agonist-free alpha-2a receptor equally well. These findings indicate that following the binding of agonists to the alpha-2a receptor, G(i-alpha) and G(o-alpha), remain physically associated with the receptor but either the conformation of G(alpha) linked to the receptor or the conformation of the receptor itself is modified such that the epitope for the C-terminal directed anti-G(i-alpha) and anti-G(o-alpha) antisera are not accessible. These agonist-induced conformational changes in the alpha-2a receptor-G(alpha) complex may be important for the activation of the G protein and the stimulation of the alpha-2a receptor signal transduction pathway.