Differential ligand efficacy at h5-HT 1A receptor-coupled G-protein subtypes: a commentary

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Activation of heterotrimeric G-protein involves the exchange by α subunits of GDP for GTP. The binding of the hydrolysis-resistant GTP analogue, guanosine-5′- O-(3-[ 35S]thio)-triphosphate ([ 35S]GTPγS), provides a measure of agonist, antagonist and inverse agonist actions at G-protein-coupled receptors. However, classical [ 35S]GTPγS binding methods do not distinguish among the subtypes of G-proteins activated. This is a limitation in view of (i) promiscuity of receptor coupling to multiple families of G-proteins, and (ii) the differential influence of ligands on distinct signal transduction pathways. A recent development of [ 35S]GTPγS binding methodology employing antibody capture and scintillation proximity assays (SPA) permits the targeting of specific Gα subtypes in both recombinant and native tissues. When applied to human serotonin 1A receptor (h5-HT 1A) expressed in Chinese hamster ovary (CHO) cells, this methodology revealed surprising patterns of Gα i3 subunit activation. For example, low but not high concentrations of high-efficacy h5-HT 1A agonists direct receptor signalling to Gα i3. In contrast, partial agonists favour h5-HT 1A receptor signalling to Gα i3 over a wide concentration range. Further, alterations in buffer sodium concentration reversed these actions of agonists: stimulation of Gα i3 activation was observed at high sodium concentration, but inhibition was observed at low sodium concentration, suggestive of protean efficacy, i.e. a switch from positive to negative efficacy dependent on receptor tone. These results indicate that complex changes in both magnitude and direction of response to receptor ligands can occur for specific G-protein subtypes. Interestingly, the inverse agonist, spiperone, inhibited constitutive h5-HT 1A receptor-mediated Gα i3 activation under all conditions tested, consistent with the hypothesis that it selectively stabilises distinct receptor conformation(s). Further, whereas classical [ 35S]GTPγS binding assays in CHO cell membranes failed to demonstrate negative efficacy for the neuroleptic, haloperidol, this compound exhibited robust inverse agonism for the activation of Gα i3 subunits. These data suggest that haloperidol may selectively inhibit constitutive activation of this G-protein subtype and, thus, exhibit inverse agonist-mediated trafficking of receptor signalling.