AbstractIn HEK-293T cells transiently transfected with the human histamine H3receptor (hH3R), we studied the effect of over-expressing the human RGS9-2 protein on H3R-mediated stimulation of [35S]-GTPγS binding and inhibition of forskolin-induced cAMP formation. Maximal specific binding (Bmax) of [3H]-N-methyl-histamine to cell membranes was 468 ± 12 and 442 ± 38 fmol/mg protein for HEK-293T-hH3R and HEK-293T-hH3R/hRGS9-2 cells, respectively, with dissociation constants (Kd) 2.57 nM and 3.38 nM. The H3R agonist immepip stimulated [35S]-GTPγS binding with similar potency and efficacy (Emax146.3 ± 4.4 % and 150.0 ± 5.3 % of basal, pEC508.57 ± 0.26 and 9.00 ± 0.33, respectively), but was significantly less efficacious to inhibit forskolin-induced cAMP accumulation in HEK-293T-hH3R/hRGS9 cells (−19.2 ± 5.3 versus −37.7 ± 5.1 % in HEK-293T-hH3R cells) with no significant difference in potency (pEC509.60 ± 0.14 and 9.07 ± 0.29, respectively). These results indicate that in HEK-293T cells hRGS9-2 regulates hH3R445signaling downstream G protein activation.