Direct measurements of in situ interactions of rat brain opioid receptors with the guanine nucleotide-binding protein Go.
The interactions of rat brain cortical opioid receptors with the guanine nucleotide-binding protein (G protein) Go were probed in membranes by examining the ability of selective antipeptide anti-G protein antisera to disrupt receptor-G protein interactions. This was measured both by antibody-induced alterations in the characteristics of agonist binding to mu and delta receptor binding sites and by antibody attenuation of opioid stimulation of high affinity GTPase activity. Antisera to the amino-terminal 16 amino acids (ON1), amino acids 22-35 (IM1), and the carboxyl-terminal decapeptide (OC2) of forms of Go alpha were able to selectively immunoprecipitate Go from rat cortical membranes. Both antisera OC2 and ON1 were able to immunoprecipitate Go alpha quantitatively. Preincubation of rat cortical membranes with an IgG fraction isolated from antiserum OC2 was able to produce a marked reduction in the ability of the synthetic enkephalin [D-Ala2,D-Leu5] enkephalin (DADLE) (which interacts with delta and mu but not significantly with kappa receptors) to displace specific binding of [3H] diprenorphine (which binds to all of these sites), demonstrating a clear interaction of the mu and delta receptors with one or more variants of Go. An IgG fraction from antiserum ON1 was able to mimic this effect, suggesting that the amino-terminal region of G protein alpha subunits also plays a role in receptor-G protein interactions. In contrast, an IgG fraction from antiserum IM1 was unable to alter the characteristics of DADLE displacement of [3H] diprenorphine binding. Similarly, an antiserum (SG1) directed against the carboxyl-terminal decapeptide common to the alpha subunits of Gi1 and Gi2 was unable to reduce the affinity of DADLE binding to opioid receptors. Use of antiserum OC2 in experiments that allowed pharmacological examination of only the mu-opioid receptor provided independent evidence for the interaction of this receptor site with Go.
Institute of Biology, National Center for Scientific Research, Demokritos, Athens, Greece.
Z
Z
Georgoussi
C
C
Carr
G
G
Milligan
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D004174:Diprenorphine / Q000378:metabolism; D016308:Enkephalin, Leucine-2-Alanine / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D007106:Immune Sera; D007074:Immunoglobulin G; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011869:Radioligand Assay; D051381:Rats; D011957:Receptors, Opioid / Q000378:metabolism*
D007106:Immune Sera; D007074:Immunoglobulin G; D011957:Receptors, Opioid; D004174:Diprenorphine; D016308:Enkephalin, Leucine-2-Alanine; D019204:GTP-Binding Proteins