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β3-Adrenoceptor desensitisation in CHO cells: comparison of cAMP and ERK signalling
Introduction: β3-Adrenoceptors (B3AR) classically couple to cAMP formation but also to alternative pathways including phosphorylation of extracellular signal-regulated kinases (ERK). We have compared desensitization of cAMP accumulation and ERK phosphorylation in Chinese Hamster ovary (CHO) cells stably transfected with human B3AR.
Method: Cell culture and cAMP experiments were performed as reported previously1, except that cAMP quantification was performed with the AlphaScreen assay (Perkin-Elmer, Rodgau, Germany). ERK phosphorylation was assessed using the AlphaLISA SureFire Ultra assay (Perkin-Elmer). Sample size was pre-specified as n=8. cAMP data are expressed as geometric means of fmol/25 μl, pERK as means of % of matched basal. Desensitization was assessed as changes in Emax and pEC50 based on paired t-tests with P<0.05.
Results: B3AR ligands (10 μM each) stimulated cAMP accumulation with a rank order of isoprenaline (255) ≈ L755,507 (247) > CL316,243 (209) > L748,337 (120) ≈ SR59,230 (108). Pre-treatment with pertussis toxin (PTX; 100 ng/ml for 24 h) did not affect the cAMP response to any ligand. Pre-treatment (10 μM for 24 h) with isoprenaline desensitized the concentration-response curves for the freshly added ligand similar to previous findings in HEK cells1 whereas pre-treatment with L748,337 had little effect. Ligands stimulated ERK phosphorylation with a rank order of L755,507 (151%) ≈ CL316,243 (149%) ≈ isoprenaline (148%) > SR59,230 (137%) > L748,337 (122%). PTX markedly lowered basal pERK, and enhanced all agonist responses relative to this lowered basal. Pretreatment with isoprenaline lowered basal, reduced potency by 0.8 log units and increased Emax relative to the lowered basal (336% to 424%). In contrast, L748,337 did not change basal pERK or pEC50 but reduced Emax (171% to 146%). Neither changed total ERK.
Conclusion: We conclude that cAMP formation in CHO cells is desensitized by the full agonist isoprenaline but not the partial agonist L748,337. In contrast, pERK responses were affected in a qualitatively different manner by pre-treatment with isoprenaline and L755,507. This appears to be the first demonstration of desensitization of pERK responses for a Gs-coupled receptor.
1. Michel-Reher MB et al. (2013). Naunyn-Schmiedebergs Arch Pharmacol 386: 843-851