A Full Pharmacological Analysis of the Three Turkey β-Adrenoceptors and Comparison with the Human β-Adrenoceptors

Abstract

BackgroundThere are three turkey β-adrenoceptors: the original turkey β-adrenoceptor from erythrocytes (tβtrunc, for which the X-ray crystal structure has recently been determined), tβ3C and tβ4C-receptors. This study examined the similarities and differences between these avian receptors and mammalian receptors with regards to binding characteristics and functional high and low affinity agonist conformations.Methodology/Principal FindingsStable cell lines were constructed with each of the turkey β-adrenoceptors and 3H-CGP12177 whole cell binding, CRE-SPAP production and 3H-cAMP accumulation assays performed. It was confirmed that the three turkey β-adrenoceptors are distinct from each other in terms of amino acid sequence and binding characteristics. The greatest similarity of any of the turkey β-adrenoceptors to human β-adrenoceptors is between the turkey β3C-receptor and the human β2-adrenoceptor. There are pharmacologically distinct differences between the binding of ligands for the tβtrunc and tβ4C and the human β-adrenoceptors (e.g. with CGP20712A and ICI118551). The tβtrunc and tβ4C-adrenoceptors appear to exist in at least two different agonist conformations in a similar manner to that seen at both the human and rat β1-adrenoceptor and human β3-adrenoceptors. The tβ3C-receptor, similar to the human β2-adrenoceptor, does not, at least so far, appear to exist in more than one agonist conformation.Conclusions/SignificanceThere are several similarities, but also several important differences, between the recently crystallised turkey β-adrenoceptor and the human β-adrenoceptors. These findings are important for those the field of drug discovery using the recently structural information from crystallised receptors to aid drug design. Furthermore, comparison of the amino-acid sequence for the turkey and human adrenoceptors may therefore shed more light on the residues involved in the existence of the secondary β-adrenoceptor conformation.

Pharmacological Analysis and Structure Determination of 7-Methylcyanopindolol–Bound β1-Adrenergic Receptor

“…4). As previously observed (Baker, 2010a), cyanopindolol elicited a biphasic response curve, with the maximum response reaching 31.8 6 1.1% (n 5 4) of the response elicited by the full agonist isoprenaline (Table 2). In contrast, 7-methylcyanopindolol gave a response that reached a maximum of 2.3 6 0.3% (n 5 4) of that attained by isoprenaline.…”

“…Pharmacological studies were conducted in Chinese hamster ovary (CHO) cells stably expressing either the turkey b 1 AR [receptor expression level 148 fmol/mg protein, defined as tßtrunc by Baker (2010a)] or the human b 2 -adrenoceptor (CHO-hb2) (receptor expression level 466 fmol/mg protein) (Baker et al, 2003). Both of these cell lines also contained the stably transfected reporter gene containing six cAMP response elements upstream of a secreted placental alkaline phosphatase reporter gene (CRE-SPAP).…”

“…2. The K D values for 3 H-CGP12177 were 0.42 nM at the turkey b 1 AR (Baker, 2010a) and 0.17 nM at the human b 2 AR (Baker, 2010b).…”

“…After 15 minutes, 10 ml of ligand was added to each well and incubated for 5 hours (37°C, 5% CO 2 ). The reaction was terminated by the addition of 50 ml of concentrated HCl per well, the plates were frozen and thawed, and 3 H-cAMP separated from other 3 H-nucleotides by sequential Dowex and alumina column chromatography, as previously described (Baker, 2010a). Basal activity and a positive control (response to 10 mM of isoprenaline) were included in all plates for every experiment.…”

Identification of Key Residues in Transmembrane 4 Responsible for the Secondary, Low-Affinity Conformation of the Human β1-Adrenoceptor

“…CGP12177 is a conventional partial agonist at the human b 2 and turkey b 3 C adrenoceptors, (i.e., with similar K B values and EC 50 values) suggesting interaction via a single conformation (Pak and Fishman, 1996;Baker et al, 2002;Baker, 2010b).…”