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

Abstract

1 This study characterizes the mouse b 3a -adrenoceptor (AR) and the splice variant of the b 3 -AR (b 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 71 ) were determined by saturation binding with [ 125 I]-(7)-cyanopindolol. Competition binding studies showed no signi®cant di erences in a nity of b-AR ligands for either receptor. 3 Several functional responses of each receptor were measured, namely extracellular acidi®cation rate (EAR; cytosensor microphysiometer), cyclic AMP accumulation, and Erk1/2 phosphorylation. The b 3 -AR agonists BRL37344, CL316243, GR265162X, L755507, SB251023, the non-conventional partial b-AR agonist CGP12177 and the b-AR agonist (7)-isoprenaline caused concentrationdependent increases in EAR in cells expressing either splice variant. CL316243 caused concentrationdependent 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 b 3b -AR but not in cells expressing the b 3a -AR at all levels of receptor expression. 5 CL316243 increased Erk1/2 phosphorylation with pEC 50 values and maximum responses that were not signi®cantly di erent in cells expressing either splice variant. Erk1/2 phosphorylation was insensitive to PTX or H89 (PKA inhibitor) but was inhibited by LY294002 (PI3Kg 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 b 3a -or b 3b -AR transfected cells. 7 These results suggest that in CHO-K1 cells, the b 3b -AR, can couple to both G s and G i to stimulate and inhibit cyclic AMP production respectively, while the b 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.

Functional and Molecular Characterization of β-Adrenoceptors in the Internal Anal Sphincter

“…Second, ␤ 3 -AR in the IAS smooth muscle may have a large number of spare receptors. Third, ␤ 3 -AR may represent a heterogeneous population such as ␤ 3a -and ␤ 3b -ARs, as suggested by the recent studies in CHO (Hutchinson et al, 2002). Furthermore, the activation and signal transduction of such a ␤ 3a -and ␤ 3b -AR complex may prevent the full potency of the ␤ 3 -AR agonist.…”

Tools to study β3-adrenoceptors

“…Studies including only β 3 -adrenoceptors reported BRL 37,344 to be a full agonist for cAMP accumulation in intact CHO cells at human and mouse but a partial agonist at rat β 3 -adrenoceptors (Blin et al 1994;Liggett 1992). BRL 37,344 has similar affinity for both splice variants of the murine β 3 -adrenoceptor, but its effects, similar to that of many other agonists, are somewhat lower at the β 3b -compared with the β 3a -adrenoceptor, as the former couples to both G s and G i proteins (Hutchinson et al 2002). Given that the selectivity of BRL 37,344 for β 3 -relative to other β-adrenoceptor subtypes is only moderate in binding studies, the question as to whether it has intrinsic efficacy at β 1 -and/or β 2 -adrenoceptors becomes of major importance to determine its usefulness in functional studies.…”

“…Moreover, the organization of the β 3 -adrenoceptor gene differs between species, with, e.g., the human gene having only two exons (the second one comprising only the final six amino acids), whereas the rodent gene has three exons (although the third does not code for any amino acid residues) . Finally, the murine receptor has splice variants, which are differentially expressed in tissues (Evans et al 1999) and couple to Naunyn-Schmiedeberg's Arch Pharmacol (2007) 374:385-398distinct signaling pathways (Hutchinson et al 2002;.…”

Evidence for Pleiotropic Signaling at the Mouse β3-Adrenoceptor Revealed by SR59230A [3-(2-Ethylphenoxy)-1-[(1,S)-1,2,3,4-tetrahydronapth-1-ylamino]-2S-2-propanol Oxalate]

“…In the case of the mouse ␤ 3 -adrenoceptor, alternative splicing results in the expression of two splice variants, the ␤ 3a -and ␤ 3b -adrenoceptor (Evans et al, 1999). Functional studies of the ␤ 3a -and ␤ 3b -adrenoceptor expressed in Chinese hamster ovary (CHO-K1) cells, using either extracellular acidification rate (ECAR) in the cytosensor microphysiometer or cAMP generation as bioassays, have shown that the ␤ 3a -and ␤ 3b -adrenoceptor couple to Gs, whereas the ␤ 3b -adrenoceptor also couples to Gi (Hutchinson et al, 2002). Both splice variants also couple to extracellular signal-related kinases 1 and 2 (ERK1/2) by a mechanism that does not involve either the generation of cAMP or activation of Gi, suggesting that additional pathways can be activated that are not linked sequentially to the classic pathways (Hutchinson et al, 2002).…”

“…Functional studies of the ␤ 3a -and ␤ 3b -adrenoceptor expressed in Chinese hamster ovary (CHO-K1) cells, using either extracellular acidification rate (ECAR) in the cytosensor microphysiometer or cAMP generation as bioassays, have shown that the ␤ 3a -and ␤ 3b -adrenoceptor couple to Gs, whereas the ␤ 3b -adrenoceptor also couples to Gi (Hutchinson et al, 2002). Both splice variants also couple to extracellular signal-related kinases 1 and 2 (ERK1/2) by a mechanism that does not involve either the generation of cAMP or activation of Gi, suggesting that additional pathways can be activated that are not linked sequentially to the classic pathways (Hutchinson et al, 2002). Therefore, it is possible that the study of a variety of ␤ 3 -adrenoceptor ligands may reveal differences in intrinsic activity (IA) at the different signaling pathways and thus shed light on the mechanisms involved.…”