DIRECT MEASUREMENTS OF IN-SITU INTERACTIONS OF RAT-BRAIN OPIOID RECEPTORS WITH THE GUANINE-NUCLEOTIDE-BINDING PROTEIN-G(O)

The interactions of rat brain cortical opioid receptors with the guani ne nucleotide-binding protein (G protein) G(o) were probed in membrane s by examining the ability of selective antipeptide anti-G protein ant isera to disrupt receptor-G protein interactions. This was measured bo th by antibody-induced alterations in the characteristics of agonist b inding to mu and delta receptor binding sites and by antibody attenuat ion of opioid stimulation of high affinity GTPase activity. Antisera t o the amino-terminal 16 amino acids (ON1), amino acids 22-35 (IM1), an d the carboxyl-terminal decapeptide (OC2) of forms of G(oalpha) were a ble to selectively immunoprecipitate G(o) from rat cortical membranes. Both antisera OC2 and ON1 were able to immunoprecipitate G(oalpha) qu antitatively. Preincubation of rat cortical membranes with an IgG frac tion isolated from antiserum OC2 was able to produce a marked reductio n in the ability of the synthetic enkephalin [D-Ala2,D-Leu5] enkephali n (DADLE) (which interacts with delta and mu but not significantly wit h kappa receptors) to displace specific binding of [H-3] diprenorphine (which binds to all of these sites), demonstrating a clear interactio n of the mu and delta receptors with one or more variants of G(o). An IgG fraction from antiserum ON1 was able to mimic this effect, suggest ing that the amino-terminal region of G protein alpha subunits also pl ays a role in receptor-G protein interactions. In contrast, an IgG fra ction from antiserum IM1 was unable to alter the characteristics of DA DLE displacement of [H-3] diprenorphine binding. Similarly, an antiser um (SG1) directed against the carboxyl-terminal decapeptide common to the alpha subunits of G(i1) and G(i2) was unable to reduce the affinit y of DADLE binding to opioid receptors. Use of antiserum OC2 in experi ments that allowed pharmacological examination of only the mu-opioid r eceptor provided independent evidence for the interaction of this rece ptor site with G(o).