Immunoprecipitation of high-affinity, guanine nucleotide-sensitive, solubilized mu-opioid receptors from rat brain: Coimmunoprecipitation of the G proteins G(alpha o), G(alpha i1), and G(alpha i3)

Antibodies directed against the C-terminal and the N-terminal regions of th e mu-opioid receptor were generated to identify the G proteins that coimmun oprecipitate with the mu receptor. Two fusion proteins were constructed: On e 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 dir ected against both fusion proteins were capable of immunoprecipitating simi lar to 70% of solubilized rat brain mu receptors as determined by [H-3][D-A la(2),N-Me-Phe(4),Gly-ol(5)]-enkephalin ([H-3]DAMGO) saturation binding. Th e 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 pell et. The ratio of G proteins to mu receptors in the immunoprecipitated mater ial was 1:1. When the material immunoprecipitated with affinity-purified an tibody was screened for the presence of G protein ac subunits, it was deter mined that G(alpha o), G(alpha i1), G(alpha i3), and to a lesser extent G(a lpha i2), but not G(alpha s) or G(alpha q/11), were coimmunoprecipitated wi th the CL receptor. Inclusion of GTP gamma S during the immunoprecipitation process abolished the coimmunoprecipitation of G proteins.