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

Abstract

Background and purpose

β₃-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 β₃-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 isoprenaline-induced 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 β₃-adrenoceptor when expressed at near physiological levels in a human cell line.

Introduction

β₃-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 β₃-adrenoceptors in adipocytes is less prominent in humans than in rodents (Arch, 2008), they play a major role 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 β₃-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 disease associations would be consistent with the idea that the 64R SNP yields a hypofunctional variant of the β₃-adrenoceptor.

To test this possibility, mechanistic studies have been performed. In some studies the ability of β₃-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 β₃-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, we have recently detected a hitherto unknown SNP in the transmembrane region 6 of the human β₃-adrenoceptor, L306F (data not shown). This SNP is of interest because it resides directly next to an amino acid which is conserved in all nine adrenoceptors and forms part of the binding pocket for catecholamines (Swaminath et al., 2005). It is possible that some of these polymorphisms form haplotypes which have a more consistent effect on receptor function than either polymorphism alone.

Against this background, the present study was designed to investigate the following questions: Does the W64R SNP affect β₃-adrenoceptor function when expressed in a human cell? Do any of the more recently discovered T265M and L306F polymorphisms affect receptor function? Is any such alteration specific for a certain chemical class of agonists? Do combinations of these three coding SNPs have effects which extend beyond those of a single SNP in isolation? To address these questions we have used the endogenous agonist noradrenaline, the prototypical agonist isoprenaline, two β₃-selective agonists currently in clinical development, (R)-2-(2-aminothiazol-4-yl)-4′-{2-[(2-hydroxy-2-phenylethyl)amino]ethyl} acetanilide (YM 178) (Takasu et al., 2007; Chapple et al., 2008) and 4′-(2-{[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino}ethyl)-3-methoxybiphenyl-4-carboxylic acid hydrochloride (FK 4664) (Imanishi et al., 2008), and (-)-4-(3-tert-butylamino-2-hydroxypropoxy)-benzimidazol-2-one (CGP 12 177), an agonist from a markedly different chemical class than the other compounds.

Methods

Site-directed mutagenesis

pcDNA3.1 (+) containing the entire coding region of the human β₃-adrenoceptor gene was purchased from UMR cDNA Resource Center (Rolla, MO, USA). Site-directed mutagenesis was performed with the QuikChange® Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) according to manufacturer guidelines with small modifications, that is, 4% DMSO was added to the PCR solution and reactions were carried out at 66°C on a Biometra T1 thermocycler (Göttingen, Germany). In some cases this procedure was repeated to introduce a second mutation into the receptor. The primers used for the mutagenesis PCR (Table 1) were constructed by use of Primer3 (Rozen and Skaletsky, 2000) and were purchased from Biolegio (Nijmegen, the Netherlands). The sequence identity of the obtained plasmid was checked by Baseclear (Leiden, the Netherlands).

Table 1.

Primers used for the mutagenesis PCR

W64R – forward	5′-CATCGTGGCCATCGCCCGGACTCCGAGACTCC-3′
W64R – reverse	5′-GGAGTCTCGGAGTCCGGGCGATGGCCACGATG-3′
T265M – forward	5′-CCCCGGTGGGGATGTGCGCTCCGCC-3′
T265M – reverse	5′-GGCGGAGCGCACATCCCCACCGGGG-3′
L306F – forward	5′-CTCTCTGCTGGTTCCCCTTCTTTCTGG-3′
L306F – reverse	5′-CCAGAAAGAAGGGGAACCAGCAGAGAG-3′

Transfection and cell culture

Human embryonic kidney (HEK) 293 cells were stably transfected with human WT and mutated β₃-adrenoceptors using a pcDNA3.1 (+) vector. Transfection was performed by calcium phosphate co-precipitation according to Graham and van der Eb (1973). Cells were grown and passaged in an atmosphere of 5% CO₂/95% air at 37°C in a 1:1 mixture of Dulbecco's Modified Eagle's Medium with nutrient mixture F12 (DMEM/F12) supplemented with 10% heat inactivated foetal calf serum and penicillin (100 units mL⁻¹) and streptomycin (100 µg mL⁻¹). To maintain selection pressure, the antibiotic geneticin (400 µg mL⁻¹) was added to all growing cells, but was not present during the experiments. The identity of all cell lines was confirmed by sequencing.

Radioligand binding studies

Cells at approximately 80% confluence were harvested by scraping the culture flasks with a cell scraper, washed twice by centrifugation at 200× g and then homogenized in ice-cold buffer (50 mmol·L⁻¹ Tris, 0.5 mmol·L⁻¹ EDTA, pH 7.5) with an Ultra-Turrax (Janke & Kunkel, Staufen, Germany) for 10 s at full speed and then twice for 20 s each at 2/3 speed. The homogenates were centrifuged for 20 min at 50 000× g at 4°C. The pellets were resuspended in buffer and stored at −80°C. Protein content was measured according to Bradford (1976), using bovine IgG as the standard.

β₃-Adrenoceptor density in the membrane preparation was determined by [¹²⁵I]-iodocyanopindolol ([¹²⁵I]-CYP) saturation binding experiments using approximately 15 µg protein per assay and eight radioligand concentrations as previously described with definition of non-specific binding by 100 µmol·L⁻¹ isoprenaline (Niclaußet al., 2006). Clones expressing ≈100 fmol mg protein⁻¹ of the receptor were used for all subsequent experiments. In competition binding experiments a radioligand concentration of 500 pmol·L⁻¹ and four competitor concentrations per log unit of concentration increment were used. The buffer for the agonist competition binding experiments had been supplemented with 5 mmol·L⁻¹ MgCl₂.

Cyclic AMP studies

Cells stably expressing the WT or mutated β₃-adrenoceptor were pre-incubated for 1 h in medium without serum. Thereafter, they were detached from the surface using enzyme free cell dissociation buffer and washed once with Hank's balanced salt solution (HBSS). The cells were resuspended in HBSS supplemented with 5 mmol·L⁻¹ HEPES and 0.05% bovine serum albumin. The stimulation mixture contained in a twofold concentration the β₃-adrenoceptor agonists, the non-specific cAMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX, 100 µmol·L⁻¹) and 4-[(3-butoxy-4-methoxyphenyl)-methyl]-2-imidazolidinone (RO 20-1724, 100 µmol·L⁻¹), a specific type 4 phosphodiesterase inhibitor. Cells were added to the stimulation mixture 1:1 in a 384 well optiplate (625 cells per well in a total volume of 10 µl) and stimulated for 30 min at room temperature. Detection was performed as previously described (Jongsma et al., 2006) using the LANCE® cAMP 384 Kit (Perkin Elmer, Zaventem, Belgium) and measured after 3 h on a Victor plate reader (Wallac, Perkin-Elmer, Zaventem, Belgium).

Data analysis

All data shown are mean values ± SEM. In the cAMP experiments sigmoidal curves were fitted to the experimental data from each experiment to calculate pEC₅₀ and E_max, the latter being expressed as increase in cAMP accumulation over basal values as determined in that experiment. Saturation and competition radioligand binding experiments were analysed by fitting rectangular hyperbolic and sigmoidal functions, respectively, to the experimental data. The statistical significance of group differences was assessed by two-tailed t-tests or one-way analysis of variance as indicated, and P < 0.05 was considered significant. All curve fitting and statistical calculations were performed using the Prism programme (Graphpad Software, San Diego, CA, USA). The receptor nomenclature used in this article follows the recommendations of the British Journal of Pharmacology (Alexander et al., 2008).

Materials

Untransfected HEK293 cells were kindly provided by Dr M. Schmidt (Groningen, the Netherlands). [¹²⁵I]-CYP (2220 Ci mmoL⁻¹) was obtained from Amersham Biosciences (Little Chalfont, Buckinghamshire, UK). DMEM/F12, foetal calf serum, penicillin/streptomycin, and geneticin, enzyme free cell dissociation buffer were from Gibco (via Invitrogen, Breda, the Netherlands). CGP 12 177, HEPES, IBMX, isoprenaline, noradrenaline and RO 20-1724 were purchased from Sigma Aldrich (Zwijndrecht, the Netherlands). YM 178 (now known as mirabegron) and FK 4664 were from Astellas Pharma Europe B.V. (Leiderdorp, the Netherlands).

Results

We initially screened multiple clones for each of the six genotypes under investigation by saturation radioligand binding. For each genotype, a clone was picked which expressed approximately 100 fmol receptor mg protein⁻¹. [¹²⁵I]-CYP had similar affinity (K_d) for all genotypes (Table 2).

Table 2.

Effect of genotype on the affinity of [¹²⁵I]-CYP in saturation binding experiments

	Kd, pmol·L−1	Bmax, fmol mg protein−1
Wild type	306 ± 49	121 ± 35
64R	312 ± 14	159 ± 35
265M	295 ± 24	107 ± 19
306F	261 ± 53	133 ± 31
64R + 265M	308 ± 17	119 ± 29
64R + 306F	279 ± 37	162 ± 59

Data are means ± SEM of 4–8 experiments for K_d (including some with clones expressing receptor densities other than 100 fmol mg protein⁻¹) and means ± SEM of three experiments for B_max. Values did not differ significantly in a one-way analysis of variance.

The basal cAMP content was about 20–50 fmol per well. None of the agonists increased cAMP accumulation in mock-transfected cells (data not shown), whereas all induced concentration-dependent cAMP accumulation in cells expressing either the WT or a mutated receptor (Fig. 1). The potency and efficacy of each agonist was similar in WT and all mutated receptors (Fig. 1, Table 3). Consistently across all genotypes the order of potency was YM 178 > isoprenaline ≈ noradrenaline > FK 4664 > CGP 12 177, whereas the order of efficacy was isoprenaline ≈ noradrenaline > YM 178 > FK 4664 ≥ CGP 12 177 (Table 3). As CGP 12 177 has been reported to be a partial agonist in several systems (Vrydag and Michel, 2007), a maximally effective concentration of isoprenaline (10 µmol·L⁻¹) was included in each experiment. Relative to this isoprenaline response, the efficacy of CGP 12 177 was 74 ± 9%, 73 ± 8%, 77 ± 10%, 74 ± 9%, 77 ± 9%, 73 ± 3% for WT and the 64R, 265M, 306F, 64R + 265M and 64R + 306F variants respectively (P < 0.05 for all groups except 64R and 64R + 265M). Taken the cAMP response to 10 µmol·L⁻¹ isoprenaline from these and the concentration-response experiments into account (n = 11–15), again there was no significant different among genotypes (data not shown).

Figure 1.

Effect of genotype on cAMP accumulation induced by (A) noradrenaline, (B) isoprenaline, (C) YM 178, (D) FK 4664 and (E) CGP 12 177. Data are means ± SEM of 3–9 experiments, and a quantitative analysis of the data is shown in Table 3.

Table 3.

Effect of genotype on agonist efficacy (E_max) and potency (pEC₅₀) for stimulating cAMP accumulation

	Noradrenaline		Isoprenaline		YM 178		FK 4664		CGP 12 177
	Emax	pEC50	Emax	pEC50	Emax	pEC50	Emax	pEC50	Emax	pEC50
Wild type	786 ± 236	7.89 ± 0.12	790 ± 212	7.95 ± 0.05	668 ± 168	9.03 ± 0.20	567 ± 146	7.01 ± 0.21	537 ± 41	6.68 ± 0.09
64R	867 ± 296	7.88 ± 0.19	697 ± 194	8.01 ± 0.06	621 ± 133	8.96 ± 0.04	550 ± 122	7.28 ± 0.25	519 ± 66	6.62 ± 0.12
265M	800 ± 37	7.96 ± 0.06	730 ± 91	7.95 ± 0.02	559 ± 121	9.10 ± 0.06	616 ± 46	7.01 ± 0.05	570 ± 92	6.72 ± 0.08
306F	832 ± 238	7.98 ± 0.07	817 ± 91	8.00 ± 0.02	576 ± 152	9.02 ± 0.07	479 ± 105	7.02 ± 0.02	624 ± 92	6.49 ± 0.14
64R + 265M	789 ± 238	7.94 ± 0.04	726 ± 96	8.00 ± 0.03	567 ± 172	9.13 ± 0.11	490 ± 119	7.12 ± 0.04	566 ± 35	6.49 ± 0.06
64R + 306F	803 ± 142	7.89 ± 0.02	745 ± 95	7.95 ± 0.01	659 ± 146	9.15 ± 0.04	611 ± 44	7.15 ± 0.06	569 ± 54	6.56 ± 0.07

Data are mean ± SEM of E_max (fmol cAMP formed per well) and pEC₅₀ values as measured in 3–9 experiments. A graphical representation of the data is shown in Figure 1. Note that numerical differences in parameter estimates for a given agonist did not significantly differ between genotypes in a one-way analysis of variance.

To substantiate these data, we have performed agonist competition binding but have restricted those experiments to the WT, the 64R and the 306F genotypes. All five agonists exhibited steep and monophasic competition curves (Fig. 2). A quantitative analysis of these experiments revealed a rank order of potency of YM 178 ≈ CGP 12 177 > FK 4664 > isoprenaline ≥ noradrenaline (Table 4); the differences between binding and cAMP stimulation order of potency would be consistent with a lower efficacy of CGP 12 177 and perhaps FK 4664 as compared with the other agonists.

Figure 2.

Effect of genotype on competition binding by (A) noradrenaline, (B) isoprenaline, (C) YM 178, (D) FK 4664 and (E) CGP 12 177. Data are means ± SEM of 3–4 experiments, and a quantitative analysis of the data is shown in Table 4.

Table 4.

Effect of genotype on the affinity of agonists in [¹²⁵I]-CYP in competition binding experiments

	Noradrenaline	Isoprenaline	YM 178	FK 4664	CGP 12 177
Wild type	4.16 ± 0.06	4.47 ± 0.07	7.26 ± 0.09	5.35 ± 0.08	7.14 ± 0.04
64R	3.97 ± 0.10	4.23 ± 0.10	7.47 ± 0.13	5.37 ± 0.11	6.98 ± 0.08
306F	4.10 ± 0.13	4.74 ± 0.05	7.43 ± 0.06	5.37 ± 0.06	7.03 ± 0.09

Data are mean ± SEM of pK_i values as measured in 3–4 experiments. Note that noradrenaline did not always cause >50% inhibition within the tested concentration range, and hence its pK_i values must be considered estimates. A graphical representation of the data is shown in Figure 2. Note that numerical differences of pK_i values for a given agonist did not significantly differ between genotypes in a one-way analysis of variance.

Finally, we have assessed whether any of the mutations affects the susceptibility of the receptor to agonist-induced functional desensitization. For this purpose cells were exposed to 10 µmol·L⁻¹ isoprenaline or vehicle in the absence of serum for 24 h prior to the experiment. After washing of the cells with buffer, concentration-response curves for cAMP accumulation induced by freshly added agonist were obtained. Pre-incubation with 10 µmol·L⁻¹ isoprenaline reduced the maximum cAMP response to freshly added isoprenaline or YM 178 by about 50% but did not significantly affect the potency of either agonist (Table 5, Fig. 3).

Table 5.

Effect of genotype on isoprenaline-induced desensitization of agonist-induced cAMP accumulation

	Stimulation of cAMP accumulation with isoprenaline				Stimulation of cAMP accumulation with YM 178
	Vehicle treatment		Isoprenaline treatment		Vehicle treatment		Isoprenaline treatment
	Wild type	64R	Wild type	64R	Wild type	64R	Wild type	64R
Emax	671 ± 39	647 ± 35	267 ± 15a	261 ± 15a	639 ± 57	659 ± 50	247 ± 24a	239 ± 23a
pEC50	7.86 ± 0.23	7.95 ± 0.20	7.98 ± 0.05	7.92 ± 0.07	9.15 ± 0.27	8.94 ± 0.24	8.78 ± 0.38	8.68 ± 0.35

Data are mean ± SEM of E_max (fmol cAMP formed per well) and pEC₅₀ values as measured in three experiments. A graphical representation of the data is shown in Figure 3.

^aP < 0.05 in a paired two-tailed t-test as compared with vehicle treatment of the same genotype.

Figure 3.

Effect of genotype on isoprenaline-induced desensitization. Data are means ± SEM of three experiments, and a quantitative analysis of the data is shown in Table 5. ISO: cells pre-treated with 10 µmol·L⁻¹ isoprenaline for 24 h. The cAMP accumulation is induced by (A) isoprenaline and (B) YM 178.

Discussion and conclusions

Although the overall evidence from clinical association studies and from those with ex vivo samples from patients suggests that the W64R SNP of the β₃-adrenoceptor represents a hypofunctional receptor variant (Leineweber et al., 2004), attempts to link this polymorphism to alterations of receptor function have met with mixed success in assays using site-directed mutagenesis with subsequent heterologous expression. One possible explanation for this discrepancy may be a small overall effect of the 64R receptor variant, as the clinical data are not fully consistent across studies despite, on average, supporting the idea of the 64R variant being hypofunctional.

The present study has addressed several other potential explanations for such discrepancies. Specifically, we have studied not only the W64R but also two more recently discovered SNPs, T265M and L306F, and have also tested potential combinations of either occurring in association with the W64R SNP. Moreover, all of these experiments were performed in a human cell line, HEK293, in order to maximize the chance of detecting functional correlates of receptor polymorphisms. Our data demonstrate that none of the polymorphisms studied here alone or in combination significantly affects the affinity of the radioligand [¹²⁵I]-CYP in saturation binding studies. All of our experiments were performed in clones expressing a similar receptor density (Table 2) which is close to that expected to exist physiologically based upon studies with other β-adrenoceptor subtypes and that may explain minor differences in observed agonist potency and efficacy. Finally, we have studied not only the endogenous agonist noradrenaline and the prototypical synthetic agonist isoprenaline but also two β₃-selective agonists, YM 178 (Takasu et al., 2007) and FK 4664 (Imanishi et al., 2008), currently in clinical development for the treatment of the overactive bladder syndrome (Chapple et al., 2008) and additional an agonist from a very different chemical family, CGP 12 177. Taken together these factors should allow minimizing the chance of false negative functional differences.

Studies with SNPs in the human β₁-adrenoceptor have demonstrated direct effects on agonist efficacy, for example, the 389G conferring weaker signalling than the 389R WT (Leineweber et al., 2004). Our studies with β₃-adrenoceptor mutants in position 64, 265 and 306 did not reveal an impact of any of these polymorphisms on agonist potency or efficacy for stimulating cAMP accumulation. Studies in human adipocytes have reported that genotype-related differences in β₃-adrenoceptor function may be more prominent for some agonists than for others (Umekawa et al., 1999). Our studies using five different agonists found a similar lack of effect of genotype for all of them. As all previous similar studies had also investigated cAMP accumulation as the functional response to explore effects of genotype (Candelore et al., 1996; Pietri-Rouxel et al., 1997; Kimura et al., 2000; Isogaya et al., 2002), we had focussed on this response as well. However, in light of recent discussions on ligand-directed signalling of β₃-adrenoceptors (Hutchinson et al., 2006; Michel and Alewijnse, 2007; Sato et al., 2007), it should be noted that our findings based upon cAMP accumulation do not exclude distinct findings for other signal transduction pathways which have been linked to β-adrenoceptor signalling such as various types of potassium channels (Bieger et al., 2006; Ferro, 2006; Scherer et al., 2007) or NO synthase (Harmon et al., 2005); however, these signalling pathways are not active in HEK293 cells, and hence we could not evaluate them in our cell lines.

Studies with β₂-adrenoceptor polymorphisms suggest that they may differ functionally when occurring in combination as part of distinct haplotypes (Hahntow et al., 2006). While it is currently unknown whether the polymorphisms in position 64, 265 and 306 of the β₃-adrenoceptor physiologically occur in combination, our data do not support the idea that such combinations yield functional properties distinct from the isolated polymorphisms or the WT.

To further characterize possible genotype effects of agonist interaction with the β₃-adrenoceptor, we have performed agonist competition binding experiments. β₃-Adrenoceptor sequencing studies in 91 Caucasian subjects performed concomitantly with the present in vitro experiments did not detect the L265M SNP (data not shown), indicating that it may be relatively rare. Based upon this finding and the apparent lack of a functional effect of this polymorphism, our competition binding experiments were limited to those in position 64 and 306. The latter was of particular interest to us as this amino acid is conserved in all three human β-adrenoceptor subtypes and resides directly next to an amino acid conserved in all nine adrenoceptor subtypes, which is believed to play an important role in catecholamine binding (Swaminath et al., 2005). However, neither the 64R nor the 306F SNP affected the apparent affinity of any of the five agonists or the radioligand, which is an antagonist/weak partial agonist.

Findings from the β₂-adrenoceptor field demonstrate that SNPs may affect the susceptibility to undergo agonist-induced desensitization (Brodde, 2008). Previous literature data indicate that the sensitivity of β₃-adrenoceptors to undergo agonist-induced desensitization is cell type dependent with little desensitization occurring in some cells including transfected Chinese hamster ovary cells (Nantel et al., 1993; Chaudhry and Granneman, 1994; Candelore et al., 1996; Curran and Fishman, 1996). On the other hand, desensitization was observed in HEK293 cells (Chaudhry and Granneman, 1994), a finding which was confirmed in the present study. Our desensitization experiments were limited to the W64R polymorphism because we had meanwhile detected the 306F allele in less than 2% of a large Caucasian population (data not shown). Our findings show that the W64R polymorphism did not affect agonist-induced desensitization in HEK293 cells, which is in line with a previous report that it also did not affect the lack of desensitization in Chinese hamster ovary cells (Candelore et al., 1996).

Taken together our data demonstrate that three SNPs in the coding region of the human β₃-adrenoceptor gene alone or in combination have little effect on ligand recognition, function or regulation of receptor responsiveness. From a pharmacogenetic point of view, our findings with YM 178 and FK 4664, which are currently undergoing clinical development for the treatment of the overactive bladder syndrome (Chapple et al., 2008), suggest that the effectiveness of these agonists is unlikely to be affected by genotype. This finding appears important as the 64R allele was reported to be present in almost 50% of patients with the overactive bladder syndrome (Honda et al., 2006). However, it should be noted that the frequent 64R SNP is in linkage with multiple polymorphisms in the non-coding region of the gene which may have effects on expression levels (Hoffstedt et al., 1999). Such genetic effects on expression levels might explain why ex vivo studies have consistently reported the 64R allele to be hypofunctional (Hoffstedt et al., 1999; Umekawa et al., 1999). Our studies with site-directed mutagenesis of the coding region, by definition, cannot address this possibility which merits further investigation.

Glossary

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-methoxybiphenyl-4-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-4-methoxyphenyl)-methyl]-2-imidazolidinone

WT

wild type

YM 178

(R)-2-(2-aminothiazol-4-yl)-4’-{2-[(2-hydroxy-2-phenylethyl)amino]ethyl} acetanilide

Conflicts of interest

M.C.M. is a paid consultant of Astellas. The other authors report no conflict of interest.