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 Gi alpha, immunoprecipitated alpha 2a receptor-Gi alpha complexes. The immunoprecipitation was specific since it could be blocked by the peptide to which antiserum 8730 was generated. Antisera 3646 (anti-Gi alpha 1), 1521 (anti-Gi alpha 2), and 1518 (anti-Gi alpha 3) immunoprecipitated solubilized alpha 2a receptor-Gi alpha complexes, indicating that all three Gi 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 Gi 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, Gi 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-Gi 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.