Single nucleotide polymorphisms in the human β3-adrenoceptor gene: effects on binding of or responses to agonists?

Wim Vrydag, Christine A. Teitsma, Astrid E. Alewijnse, Martin C. Michel Dept. Pharmacology and Pharmacotherapy, AMC, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands

β3 -Adrenoceptors are involved in catecholamine-induced effects e.g. on adipose tissue and smooth muscles in blood vessels and the urinary bladder. Single nucleotide polymorphisms (SNPs) have been described in the corresponding gene, of which Trp64Arg has been investigated most often and was associated with increased body mass, worsened diabetic status, hypertension and bladder dysfunction in some but not all studies (Leineweber et al., 2004). An additional SNP in the 3rd intracellular loop, Thr265Met, has been found but no functional data have been reported. Finally, we have recently identified a Leu306Phe SNP which is of interest because it is located directly next to the proposed catecholamine binding site in the receptor. Therefore, we have investigated how these three SNPs and the 64Arg/265Met haplotype affect agonist-induced cAMP accumulation in transfected cells. Effects of agonist binding to the receptor were studied for selected SNPs.

The polymorphic receptors were generated by site-directed-mutagenesis and stably expressed in human embryonic kidney 293 cells. Clones expressing approximately 100 fmol receptor/mg protein were selected based upon [125I]-iodocyanopindolol saturation binding studies (90 min at 37°C, Niclauss et al., 2006). Cyclic AMP accumulation was measured during 30 min incubation at room temperature in the presence of 100μM each of the phosphodiesterase inhibitors isobutylmethylxanthine and Ro 20-1724 (4-[(3-Butoxy-4-methoxyphenyl)-methyl]-2-imidazolidinone) using the Lance® kit (Perkin Elmer). Data are means ± s.e. means of at least 3 experiments.

Wild-type as well as all four polymorphic receptors had an affinity of approximately 300pM for [125I]-iodocyanopindolol in saturation binding. In competition binding the 64Arg and 306Phe genotypes did not affect apparent affinities as compared to wild-type receptor for the agonists noradrenaline (pKi 4.16 ± 0.06 ) and isoprenaline (pKi 4.47 ± 0.06 ).

In cAMP accumulation studies with cells expressing wild-type receptor the potency (pEC50) of noradrenaline and isoprenaline were 7.94 ± 0.17 and 7.95 ± 0.08 , respectively. Relative to isoprenaline, noradrenaline was a full agonist for this response. The 64Arg, 265Met, 306Phe and 64Arg265Met variants exhibited similar potency and efficacy for both agonists.

We conclude that SNPs tested here lack major effects on β3-adrenoceptor function.

Leineweber, K et al. (2004) Naunyn-Schmiedeberg’s Arch. Pharmacol.369, 1-22
Niclauss, N et al. (2006) Naunyn-Schiedeberg’s Arch. Pharmacol.374, 99-105

Source of funding: Astellas Europe Ltd.