Do gene polymorphisms alone or in combination affect the function of human beta3-adrenoceptors?
OBJECTIVE
beta(3)-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.


METHODS
We transfected the wild-type human beta(3)-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.


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 isoprenaline-induced functional desensitization during a 24 h treatment.


CONCLUSIONS
None of the polymorphisms tested here significantly altered the interaction of isoprenaline, noradrenaline, YM 178, FK 4664 or CGP 12 177 with the human beta(3)-adrenoceptor when expressed at near physiological levels in a human cell line.
Vrydag
Wim
W
Department of Pharmacology and Pharmacotherapy, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
Alewijnse
Astrid E
AE
Michel
Martin C
MC
D000083:Acetanilides / Q000494:pharmacology; D058667:Adrenergic beta-3 Receptor Agonists; D000318:Adrenergic beta-Agonists / Q000494:pharmacology*; D001667:Binding, Competitive; D001713:Biphenyl Compounds / Q000494:pharmacology; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009638:Norepinephrine / Q000494:pharmacology; D010627:Phenethylamines / Q000494:pharmacology; D020641:Polymorphism, Single Nucleotide; D011412:Propanolamines / Q000494:pharmacology; D011869:Radioligand Assay; D022702:Receptors, Adrenergic, beta-3 / Q000235:genetics / Q000502:physiology*; D013844:Thiazoles / Q000494:pharmacology; D014162:Transfection
C540585:4'-(2-((2-(3-chlorophenyl)-2-hydroxyethyl)amino)ethyl)-3-methoxybiphenyl-4-carboxylic acid; D000083:Acetanilides; D058667:Adrenergic beta-3 Receptor Agonists; D000318:Adrenergic beta-Agonists; D001713:Biphenyl Compounds; D010627:Phenethylamines; D011412:Propanolamines; D022702:Receptors, Adrenergic, beta-3; D013844:Thiazoles; D000242:Cyclic AMP; D007545:Isoproterenol; C520025:mirabegron; C033394:CGP 12177; D009638:Norepinephrine