Bell-shaped agonist activation of 5-HT 1A receptor-coupled Gαi 3 G-proteins: Receptor density-dependent switch in receptor signaling.

A previous study observed bell-shaped concentration-response isotherms for activation of Gα G-protein subunits by high efficacy 5-HT 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 to activation of other G-proteins) but it was unclear if such effects are observed for 5-HT receptors in other cellular environments. Here, using an antibody capture-based [S]GTPγS binding assay for Gα activation, we investigated whether efficacious 5-HT 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 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 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 activation. These data indicate that Gα G-protein activation by 5-HT 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.