h5-HT 1B receptor-mediated constitutive Gα i3-protein activation in stably transfected Chinese hamster ovary cells: An antibody capture assay reveals protean efficacy of 5-HT

Abstract

1. Serotonin 5-HT 1B receptors couple to G-proteins of the Gi/o family. However, their activation of specific G-protein subtypes is poorly characterised. Using an innovative antibody capture/guanosine-5′-0-(3-[ 35S]thio)-triphosphate ([ 35S]GTPγS) binding strategy, we characterised Gα i3 subunit activation by h5-HT 1B receptors stably expressed in Chinese hamster ovary (CHO) cells. 2. The agonists, 5-HT, alniditan and BMS181,101, stimulated Gα i3, whereas methiothepin and SB224,289 behaved as inverse agonists. The selective 5-HT 1B receptor ligand, S18127, modestly stimulated Gα i3 and reversed the actions of both 5-HT and methiothepin. S18127 (1 μM) also produced parallel, dextral shifts of the 5-HT and methiothepin isotherms. 3. Isotopic dilution experiments ([ 35S]GTPγS versus GTPγS) revealed high-affinity [ 35S]GTPγS binding to Gαi3 subunits in the absence of receptor ligands indicating constitutive activity. High-affinity [ 35S]GTPγS binding was increased 2.8-fold by 5-HT with an increase in the affinity of GTPγS for Gα i3 subunits. In contrast, methiothepin halved the number of high-affinity binding sites and decreased their affinity. 4. h5-HT 1B receptor-mediated Gα i3 subunit activation was dependent on the concentration of NaCl. At 300 mM, 5-HT stimulated [ 35S]GTPγS binding, basal Gα i3 activation was low and methiothepin was inactive. In contrast, at 10 mM NaCl, basal activity was enhanced and the inverse agonist activity of methiothepin was accentuated. Under these conditions, 5-HT decreased Gα i3 activation. 5. In conclusion, at h5-HT 1B receptors expressed in CHO cells: (i) inverse agonist induced inhibition of Gα i3, and its reversal by S18127, reveals constitutive activation of this Gα subunit; (ii) constitutive Gα i3 activation can be quantified by isotopic dilution [ 35S]GTPγS binding and (iii) decreasing NaCl concentrations enhances Gα i3 activation and leads to protean agonist properties of 5-HT: that is a switch to inhibition of Gα i3.