h5‐HT 1B receptor‐mediated constitutive G α i3 ‐protein activation in stably transfected Chinese hamster ovary cells: an antibody capture assay reveals protean efficacy of 5‐HT

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‐[ 35 S]thio)‐triphosphate ([ 35 S]GTP γ S) binding strategy, we characterised G α i3 subunit activation by h5‐HT 1B receptors stably expressed in Chinese hamster ovary (CHO) cells. The agonists, 5‐HT, alniditan and BMS181,101, stimulated G α i3 , whereas methiothepin and SB224,289 behaved as inverse agonists. The selective 5‐HT 1B receptor ligand, S18127, modestly stimulated G α i3 and reversed the actions of both 5‐HT and methiothepin. S18127 (1 μ M ) also produced parallel, dextral shifts of the 5‐HT and methiothepin isotherms. Isotopic dilution experiments ([ 35 S]GTP γ S versus GTP γ S) revealed high‐affinity [ 35 S]GTP γ S binding to G α i3 subunits in the absence of receptor ligands indicating constitutive activity. High‐affinity [ 35 S]GTP γ S binding was increased 2.8‐fold by 5‐HT with an increase in the affinity of GTP γ S for G α i3 subunits. In contrast, methiothepin halved the number of high‐affinity binding sites and decreased their affinity. h5‐HT 1B receptor‐mediated G α i3 subunit activation was dependent on the concentration of NaCl. At 300 m M , 5‐HT stimulated [ 35 S]GTP γ S binding, basal G α i3 activation was low and methiothepin was inactive. In contrast, at 10 m M NaCl, basal activity was enhanced and the inverse agonist activity of methiothepin was accentuated. Under these conditions, 5‐HT decreased G α i3 activation. In conclusion, at h5‐HT 1B receptors expressed in CHO cells: (i) inverse agonist induced inhibition of G α i3 , and its reversal by S18127, reveals constitutive activation of this G α subunit; (ii) constitutive G α i3 activation can be quantified by isotopic dilution [ 35 S]GTP γ S binding and (iii) decreasing NaCl concentrations enhances G α i3 activation and leads to protean agonist properties of 5‐HT: that is a switch to inhibition of G α i3 . British Journal of Pharmacology (2003) 138 , 1077–1084. doi: 10.1038/sj.bjp.0705140