Immunoprecipitation of high-affinity, guanine nucleotide-sensitive, solubilized μ-opioid receptors from rat brain:: Coimmunoprecipitation of the G proteins Gαo, Gαi1, and Gαi3

Antibodies directed against the C-terminal and the N-terminal regions of the mu-opioid receptor were generated to identify the G proteins that coimmunoprecipitate with the mu receptor. Two fusion proteins were constructed: One contained the 50 G-terminal amino acids of the mu receptor, and the other contained 61 amino acids near the N terminus of the receptor. Antisera directed against both fusion proteins were capable of immunoprecipitating similar to 70% of solubilized rat brain mu receptors as determined by [H-3][D-Ala(2),N-Me-Phe(4),Gly-ol(5)]-enkephalin ([H-3]DAMGO) saturation binding. The material immunoprecipitated with both of the antisera was recognized as a broad band with a molecular mass between 60 and 75 kDa when screened in a western blot. Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) had an EC50 of 0.4 nM in diminishing [H-3]DAMGO binding to the immunoprecipitated pellet. The ratio of G proteins to mu receptors in the immunoprecipitated material was 1:1. When the material immunoprecipitated with affinity-purified antibody was screened for the presence of G protein ac subunits, it was determined that G(alpha o), G(alpha i1), G(alpha i3), and to a lesser extent G(alpha i2), but not G(alpha s) or G(alpha q/11), were coimmunoprecipitated with the CL receptor. Inclusion of GTP gamma S during the immunoprecipitation process abolished the coimmunoprecipitation of G proteins.