Bell-shaped agonist activation of 5-HT_1A receptor-coupled Gαi₃ G-proteins: Receptor density-dependent switch in...

A previous study observed bell-shaped concentration-response isotherms for activation of Gα_i3 G-protein subunits by high efficacy 5-HT_1A receptor agonists in a Chinese hamster ovary (CHO) cell line expressing high levels of these receptors. This suggested that a signaling switch took place in that cell line (from Gα_i3 to activation of other G-proteins) but it was unclear if such effects are observed for 5-HT_1A receptors in other cellular environments. Here, using an antibody capture-based [³⁵S]GTPγS binding assay for Gα_i3 activation, we investigated whether efficacious 5-HT_1A receptor agonists (5-HT, F13714, befiradol, NLX-101), prototypical agonists ((+) and (-)8-OH-DPAT), and partial agonist, antagonists, inverse agonists (pindolol, WAY100635, spiperone) produced similar effects on 5 cell lines expressing different levels of human 5-HT_1A receptors. In membranes from cell lines (HeLa, C6-glia and CHO-low) expressing moderate receptor levels (between 1 and 4 pmol/mg of protein), 5-HT, F13714, befiradol and NLX-101 elicited classical sigmoid concentration-response isotherms. In contrast, in cell lines (CHO-high, HEK-293F) expressing high receptor levels (>9 pmol/mg) these agonists elicited bell-shaped concentration-response isotherms that peaked at nanomolar-range concentrations and then returned to baseline or below. Spiperone elicited inverse agonist inhibitory sigmoid isotherms in all membrane preparations while WAY100635 was mostly 'silent' for Gα_i3 activation. The other compounds elicited diverse responses in the different cell lines suggesting that other factors, in addition to receptor expression levels, could be influencing Gα_i3 activation. These data indicate that Gα_i3 G-protein activation by 5-HT_1A receptor ligands is highly dependent on receptor expression levels and on cellular background. Moreover, the induction of bell-shape concentration-response isotherms by 5-HT and other high-efficacy agonists is consistent with a switch in signaling to other G-protein-mediated signaling cascades, possibly elicited by receptor conformational changes.