h5-HT(1B) receptor-mediated constitutive Galphai3-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-[(35)S]thio)-triphosphate ([(35)S]GTPgammaS) binding strategy, we characterised Galpha(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 Galpha(i3), whereas methiothepin and SB224,289 behaved as inverse agonists. The selective 5-HT(1B) receptor ligand, S18127, modestly stimulated Galpha(i3) and reversed the actions of both 5-HT and methiothepin. S18127 (1 micro M) also produced parallel, dextral shifts of the 5-HT and methiothepin isotherms. 3. Isotopic dilution experiments ([(35)S]GTPgammaS versus GTPgammaS) revealed high-affinity [(35)S]GTPgammaS binding to Galpha(i3) subunits in the absence of receptor ligands indicating constitutive activity. High-affinity [(35)S]GTPgammaS binding was increased 2.8-fold by 5-HT with an increase in the affinity of GTPgammaS for Galpha(i3) subunits. In contrast, methiothepin halved the number of high-affinity binding sites and decreased their affinity. 4. h5-HT(1B) receptor-mediated Galpha(i3) subunit activation was dependent on the concentration of NaCl. At 300 mM, 5-HT stimulated [(35)S]GTPgammaS binding, basal Galpha(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 Galpha(i3) activation. 5. In conclusion, at h5-HT(1B) receptors expressed in CHO cells: (i) inverse agonist induced inhibition of Galpha(i3), and its reversal by S18127, reveals constitutive activation of this Galpha subunit; (ii) constitutive Galpha(i3) activation can be quantified by isotopic dilution [(35)S]GTPgammaS binding and (iii) decreasing NaCl concentrations enhances Galpha(i3) activation and leads to protean agonist properties of 5-HT: that is a switch to inhibition of Galpha(i3).

Figure 1

Action of agonists and inverse agonists on [³⁵S]GTPγS binding to Gα_i3 subunits in CHO-h5-HT_1B cell membranes. Panel a: concentration – response isotherms of 5-HT, BMS181,101 and methiothepin. Panel b: concentration – response isotherm of alniditan, S18127 and SB224,287. Data points are means of duplicate determinations from representative experiments repeated on at least three independent occasions. Data from these experiments are summarised in Table 1.

Figure 2

Antagonism by S18127 of 5-HT-stimulated and methiothepin-inhibited [³⁵S]GTPγS binding to Gα_i3 subunits in CHO-h5-HT_1B cell membranes. Panel a: concentration – response isotherms of 5-HT and methiothepin alone or in the presence of S18127 (1 μM). Panel b: concentration-dependent reversal by S18127 of 5-HT (10 nM)-induced stimulation or methiothepin (100 nM)-induced inhibition of Gα_i3 activation. Data points are the means of duplicate determinations from representative experiments repeated on at least three independent occasions.

Figure 3

Inhibition, by isotopic dilution with GTPγS, of [³⁵S]GTPγS binding to Gα_i3 subunits in membranes of CHO cells expressing h5-HT_1B receptors. Panel a: [³⁵S]-GTPγS isotopic dilution under basal conditions (no receptor ligands) and in the presence of 5-HT (1 μM) or methiothepin (1 μM). The dotted lines indicate the HA binding component of the isotherms. Isotherms determined by nonlinear regression are biphasic (two-site fit statistically superior to a single site fit; P<0.05, F-test). Panel b: Gα_i3 subunit saturation binding isotherms derived from [³⁵S]GTPγS isotopic dilution experiments (as described in Materials and Methods). Isotherms are shown under basal conditions and in the presence of 5-HT (1 μM) or methiothepin (1 μM). Points shown are from representative experiments performed in duplicate and repeated on at least three independent occasions. Data from these experiments are summarised in Table 2.

Figure 4

Influence of NaCl concentrations on [³⁵S]GTPμS binding to Gα_i3 subunits in CHO-h5-HT_1B cell membranes. The influence of four concentrations of NaCl on the actions of a full agonist, 5-HT (panel a), and on the actions of the inverse agonist, methiothepin (panel b), are shown. B = basal binding. Data points are the means of duplicate determinations from representative experiments repeated on at least three independent occasions with similar results. Data from these experiments are summarised in Table 3.