Immunoprecipitation of High‐Affinity, Guanine Nucleotide‐Sensitive, Solubilized μ‐Opioid Receptors from Rat Brain

Antibodies directed against the C‐terminal and the N‐terminal regions of the μ‐opioid receptor were generated to identify the G proteins that coimmunoprecipitate with the μ receptor. Two fusion proteins were constructed: One contained the 50 C‐terminal amino acids of the μ 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 ∼70% of solubilized rat brain μ receptors as determined by [ 3 H][D‐Ala 2 , N ‐Me‐Phe 4 ,Gly‐ol 5 ]‐enkephalin ([ 3 H]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) (GTPγS) had an EC 50 of 0.4 n M in diminishing [ 3 H]DAMGO binding to the immunoprecipitated pellet. The ratio of G proteins to μ receptors in the immunoprecipitated material was 1:1. When the material immunoprecipitated with affinity‐purified antibody was screened for the presence of G protein α subunits, it was determined that G αo , G αi1 , G αi3 , and to a lesser extent G αi2 , but not G αs or G αq/11 , were coimmunoprecipitated with the μ receptor. Inclusion of GTPγS during the immunoprecipitation process abolished the coimmunoprecipitation of G proteins.