Do gene polymorphisms alone or in combination affect the function of human β₃‐adrenoceptors?

Abstract

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.

Functional investigation of β-adrenoceptors in human isolated detrusor focusing on the novel selective β3-adrenoceptor agonist KUC-7322

“…However, it has been reported that the human β3-AR gene is polymorphic and that the prevalence of the most common polymorphism Trp64Arg differs between ethnicities (Belfer et al 2005;Honda et al 2006;Walton et al 1995). The relevance of this differential genotype preference, however, is uncertain as it has not been consistently associated with altered agonist responses (Vrydag et al 2009). Our current findings that the potency of both isoproterenol and KUC-7322 is virtually identical in studies I (Japanese patients) and II (German patients) further support that there is no major ethnic difference for β-adrenergic bladder relaxation.…”

The Odd Sibling: Features ofβ3-Adrenoceptor Pharmacology

“…Interestingly, in cases where b 3 -adrenoceptor desensitization was observed, it often occurred at the level of receptor mRNA expression (Granneman and Lahners, 1992;Bengtsson et al, 1996;Scarpace et al, 1999) or that of signaling molecules activated by the receptor (Chambers et al, 1994;Michel-Reher and Michel, 2013), whereas downregulation of the receptor itself at the protein level has rarely been reported . The Trp64Arg polymorphism of the receptor apparently affects neither the lack of downregulation in Chinese hamster ovary cells (Candelore et al, 1996) nor the desensitization in human embryonic kidney cells (Vrydag et al, 2009). Taken together, agonist-induced b 3 -adrenoceptor desensitization can occur in some but not other cell types and tissues; however, in line with the lack of phosphorylation sites, when it does occur, it largely involves downregulation of corresponding mRNA and/or an altered expression of signaling molecules.…”

“…Re-creation of the Trp64Arg polymorphism by site-directed mutagenesis studies has also yielded inconsistent results (Vrydag et al, 2009;Engelhardt and Ahles, 2014). On the other hand, sequencing studies showed that the Trp64Arg polymorphism forms a haploblock with several polymorphisms in the noncoding part of the b 3 -adrenoceptor gene, including single nucleotide and TG dinucleotide length polymorphisms (Table 2).…”

Beta-3 Adrenoceptors as New Therapeutic Targets for Cardiovascular Pathologies

“…A missense mutation of the human ADRB3 gene replacing tryptophan with arginine at codon 64 (Trp64Arg) previously has been reported to be associated with some cardiovascular risk factors such as obesity, diabetes mellitus, and elevated blood pressure. It is important to note that in a heterologous expression system, this ß 3 -AR polymorphism failed to alter interaction of the receptor with its ligand [49]. A recent cohort study combined with a meta-analysis of previous reports does not support a role of Trp64Arg human ß 3 -AR polymorphism in coronary heart disease risk [50].…”