Receptor-mediated Gi-3 activation in mammalian and human brain membranes: Reestablishment method and its application to nociceptin/orphanin FQ opioid peptide (NOP) receptor/Gi-3 interaction.

Functional activation of heterotrimeric guanine nucleotide-binding proteins (G-proteins) via G-protein-coupled receptors (GPCRs) has been extensively explored using guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding assay. However, the conventional method is primarily applicable to Gi/o family without discrimination among G-protein subtypes. Therefore, this study aims to reestablish a novel method termed "[35S]GTPγS binding/immunoprecipitation assay" by identifying a most suitable anti-Gαi-3 antibody instead of the previously utilized, now withdrawn antibody. In the initial screening of commercially available anti-Gαi-3 antibodies, two were identified and one was selected for further investigations based on efficacy with adenosine-the most potent agonist in our previous research. After optimizing experimental conditions with rat and postmortem human brain membranes, the stimulatory effects of various agonists were evaluated. Some agonists, including nociceptin, exhibited sufficient stimulatory effects for further pharmacological characterization. Nociceptin increased [35S]GTPγS binding to Gαi-3 in a concentration-dependent manner, response that was insensitive to naloxone but potently inhibited using (±)-J-113397. The method described in this study provides a valuable strategy for determining the intrinsic efficacy of ligands at various GPCRs. This includes nociceptin/orphanin FQ opioid peptide (NOP) receptor selectively coupled to Gαi-3, providing insights into the pharmacological concept of "functional selectivity."