Mouse β3a- and β3b-adrenoceptors expressed in Chinese hamster ovary cells display identical pharmacology but utilize distinct signalling pathways

1 This study characterizes the mouse beta(3a)-adrenoceptor (AR) and the splice variant of the beta(3)-AR (beta(3b)-AR) expressed in Chinese hamster ovary cells (CHO-K1). 2 Stable clones with high (similar to 1200), medium (similar to 500) or low receptor expression (similar to 100 fmol mg protein(-1)) were determined by saturation binding with [I-125]-(-)-cyanopindolol. Competition binding studies showed no significant differences in affinity of beta-AR ligands for either receptor. 3 Several functional responses of each receptor were measured, namely extracellular acidification rate (EAR; cytosensor microphysiometer), cyclic AMP accumulation, and Erk1/2 phosphorylation. The beta(3)-AR agonists BRL37344, CL316243, GR265162X, L755507, SB251023, the non-conventional partial beta-AR agonist CGP12177 and the beta-AR agonist (-)-isoprenaline caused concentration-dependent increases in EAR in cells expressing either splice variant. CL316243 caused concentration-dependent increases in cyclic AMP accumulation and Erk1/2 phosphorylation in cells expressing either receptor. 4 PTX treatment increased maximum EAR and cyclic AMP responses to CL316243 in cells expressing the beta(3b)-AR but not in cells expressing the beta(3a)-AR at all levels of receptor expression. 5 CL316243 increased Erk1/2 phosphorylation with pEC(50) values and maximum responses that were not significantly different in cells expressing either splice variant. Erk1/2 phosphorylation was insensitive to PTX or H89 (PKA inhibitor) but was inhibited by LY294002 (P13Kgamma inhibitor), PP2 (c-Src inhibitor), genistein (tyrosine kinase inhibitor) and PD98059 (MEK inhibitor). 6 The adenylate cyclase activators forskolin or cholera toxin failed to increase Erk-1/2 levels although both treatments markedly increased cyclic AMP accumulation in both beta(3a)- or beta(3b)-AR transfected cells. 7 These results suggest that in CHO-K1 cells, the beta(3b)-AR, can couple to both G(s) and G(i) to stimulate and inhibit cyclic AMP production respectively, while the beta(3a)-AR, couples solely to G(s) to increase cyclic AMP levels. However, the increase in Erk1/2 phosphorylation following receptor activation is not dependent upon coupling of the receptors to G(i) or the generation of cyclic AMP.