Mouse β_3a and β_3b-adrenoceptors expressed in Chinese hamster ovary cells display identical pharmacology but utilize distinct signalling pathways

1. This study characterizes the mouse β_3a-adrenoceptor (AR) and the splice variant of the β₃-AR (β_3b-AR) expressed in Chinese hamster ovary cells (CHO-K1). 2. Stable clones with high (∼ 1200), medium (∼ 500) or low receptor expression (∼ 100 fmol mg protein^-1) were determined by saturation binding with [¹²⁵I]-(-)-cyanopindolol. Competition binding studies showed no significant differences in affinity of β-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 β₃-AR agonists BRL37344, CL316243, GR265162X, L755507, SB251023, the non-conventional partial β-AR agonist CGP12177 and the β-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 β_3b-AR but not in cells expressing the β_3a-AR at all levels of receptor expression. 5. CL316243 increased Erk1/2 phosphorylation with pEC₅₀ 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 (PI3Kγ 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 Erk1/2 levels although both treatments markedly increased cyclic AMP accumulation in both β_3a- or β_3b-AR transfected cells. 7. These results suggest that in CHO-K1 cells, the β_3b-AR, can couple to both G_s and G_i to stimulate and inhibit cyclic AMP production respectively, while the β_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.