Do gene polymorphisms alone or in combination affect the function of human beta3-adrenoceptors?

British Journal of Pharmacology (2009), 156, 127–134 © 2008 The Authors Journal compilation © 2008 The British Pharmacological Society All rights reserved 0007-1188/08 www.brjpharmacol.org RESEARCH PAPER Do gene polymorphisms alone or in combination affect the function of human b3-adrenoceptors? Wim Vrydag, Astrid E. Alewijnse and Martin C. Michel Department of Pharmacology and Pharmacotherapy, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands Background and purpose: b3-Adrenoceptors mediate many important physiological functions, for example, in the urinary bladder. The corresponding gene is polymorphic, and the W64R (Trp64Arg) single nucleotide polymorphism has been associated with disease states such as obesity, type 2 diabetes and bladder dysfunction. While these clinical data suggest that the 64R variant is hypofunctional, previous in vitro studies in which this variant was generated by site-directed mutagenesis and subsequent transfection have not consistently confirmed this. Experimental approach: We transfected the wild-type human b3-adrenoceptor and the 64R variant and also the more recently discovered 265M and 306F variants as well as 64R/265M and 64R/306F double mutants into human embryonic kidney cells and selected clones expressing the receptors at a density of about 100 fmol mg protein-1. Receptor activation was measured by cAMP accumulation and ligand affinity by radioligand binding. Desensitisation was assessed as alterations of cAMP responses after prolonged agonist treatment. Key results: Neither mutated receptor exhibited alterations in efficacy or potency for cAMP accumulation for any of five agonists (isoprenaline, noradrenaline, YM 178, FK 4664, CGP 12 177). In competition binding studies, the mutations did not affect the ability of any agonist to bind to the receptor. Wild-type receptors and the 64R variant exhibited similar isoprenalineinduced functional desensitization during a 24 h treatment. Conclusions and implications: None of the polymorphisms tested here significantly altered the interaction of isoprenaline, noradrenaline, YM 178, FK 4664 or CGP 12 177 with the human b3-adrenoceptor when expressed at near physiological levels in a human cell line. British Journal of Pharmacology (2009) 156, 127–134; doi:10.1111/j.1476-5381.2008.00014.x Keywords: Trp64Arg; Thr265Met; Leu306Phe; b3-adrenoceptor; polymorphism; YM 178; FK 4664; CGP 12 177 Abbreviations: CGP 12,177, (-)-4-(3-tert-butylamino-2-hydroxypropoxy)-benzimidazol-2-one; DMEM, Dulbecco’s Modified Eagle’s Medium; FK 4664, 4′-(2-{[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino}ethyl)-3-methoxybiphenyl4-carboxylic acid hydrochloride; HBSS, Hank’s balanced salt solution; HEK, human embryonic kidney; IBMX, 3-isobutyl-1-methylxanthine; [125I]-CYP, [125I]-iodocyanopindolol; RO 20-1724, 4-[(3-butoxy-4methoxyphenyl)-methyl]-2-imidazolidinone; WT, wild type; YM 178, (R)-2-(2-aminothiazol-4-yl)-4’-{2-[(2hydroxy-2-phenylethyl)amino]ethyl} acetanilide Introduction in the regulation of smooth muscle tone in the human urinary bladder (Michel and Vrydag, 2006), possibly both at the smooth muscle and the urothelial level (Otsuka et al., 2008). The human b3-adrenoceptor gene is located on chromosome 8p11–8p12 (Emorine et al., 1989). More than 10 years ago, this gene was identified as being polymorphic and the W64R (Trp64Arg) single nucleotide polymorphism (SNP) has received considerable attention (Clement et al., 1995; Walton et al., 1995). Although the existing studies are not unequivocal, the overall evidence suggests that the 64R genotype can be associated with disease states such as obesity and type 2 diabetes (Leineweber et al., 2004). A more recent report has also linked the 64R genotype to the overactive bladder syndrome (Honda et al., 2006). All of these b3-Adrenoceptors mediate effects of the endogenous catecholamines adrenaline and noradrenaline in several tissues including adipose tissue and smooth muscle where they can mediate lipolysis and thermogenesis or relaxation respectively (Rozec and Gauthier, 2006; Arch, 2008). While the role of b3-adrenoceptors in adipocytes is less prominent in humans than in rodents (Arch, 2008), they play a major role Correspondence: Professor Martin C. Michel, Department of Pharmacology and Pharmacotherapy, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, the Netherlands. E-mail: Received 16 January 2008; revised 20 August 2008; accepted 22 August 2008 128 Polymorphisms of human b3-adrenoceptors W Vrydag et al disease associations would be consistent with the idea that the 64R SNP yields a hypofunctional variant of the b3-adrenoceptor. To test this possibility, mechanistic studies have been performed. In some studies the ability of b3-adrenoceptor agonists to induce lipolysis was compared in samples obtained from subjects stratified according to genotype, and the results were compatible with the proposed hypofunction of the 64R genotype (Hoffstedt et al., 1999; Umekawa et al., 1999). On the other hand, similar studies have been performed with transfected cells in which the 64R genotype had been generated by site-directed mutagenesis. While some of these studies support the idea of the 64R variant yielding a hypofunctional receptor (Pietri-Rouxel et al., 1997; Kimura et al., 2000), such differences were not detected in other studies (Candelore et al., 1996), and some investigators have even reported an increased function for the 64R variant under certain circumstances, that is, when co-expressed with type III adenylyl cyclase (Isogaya et al., 2002). When considering these somewhat heterogeneous results, three potential explanations come to mind. First, it is possible that a given SNP will selectively affect the response to certain agonists because it has more effect on the binding pocket of a specific compound than on the general receptor function (Michel and Alewijnse, 2007). This possibility is highlighted by findings in human adipocytes (Umekawa et al., 1999). Second, the polymorphic allele 64R in humans is the wild type (WT) in almost all animal species including chimpanzees (Vrydag and Michel, 2007), but most expression studies have not been done in human cells but rather in those derived from other species. Therefore, the possibility exists that these cells do not have the correct machinery to fully translate functional effects of this polymorphism. Third, the W64R SNP is not the only polymorphism in the human b3-adrenoceptor gene. Thus, it has been shown that this SNP forms a haploblock with several polymorphisms in the non-coding region of the receptor which possibly affect transcription of the gene and/or the stability of the corresponding mRNA (Hoffstedt et al., 1999). Moreover, an additional SNP in the coding region (T265M) has been reported (Halushka et al., 1999), which may be in linkage disequilibrium with the W64R polymorphism but nothing is known about the functional properties of this SNP. Finally (...truncated)