A full pharmacological analysis of the three turkey β-adrenoceptors and comparison with the human β-adrenoceptors

Abstract

Background: There 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 findings: Stable cell lines were constructed with each of the turkey β-adrenoceptors and 3H-CGP12177 whole cell binding, CRE-SPAP production and (3)H-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/significance: There 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.

Figure 1. The amino acid sequence of the three turkey and the three human β-adrenoceptors.

Amino acid sequences shown for published receptors are taken from UniProtKB/Swiss-Prot and correspond to the translation of the coding sequence from each nucleotide receptor construct used in the present study. All receptor constructs were checked by sequencing on both strands using automated fluorescent sequencing (University of Nottingham, Nottingham, U.K.).

Figure 2. Inhibition of ³H-CGP12177 specific binding to whole cells expressing the 3 turkey β-adrenoceptors.

Inhibition of ³H-CGP12177 specific binding to whole cells by CGP20712A and ICI118551 in a) CHO tβtrunc cells, b) CHO tβ3C cells and c) CHO tβ4C cells. Non-specific binding was determined by 10 µM propranolol. The concentrations of ³H-CGP 12177 present in each case are a) 1.24 nM, b) 0.855 nM and c) 1.59 nM. Data points are mean ± s.e.mean of triplicate determinations. These single experiments are representative of a) 5, b) 9 and c) 9 separate experiments.

Figure 3. Correlation plot of the log K_D values for the turkey β-adrenoceptors compared with each other.

Ligands are labelled as full agonists if the stimulated more than 90% of the response at the human β1-adrenoceptor, as partial agonists if they stimulated 5–90% of the full response and as antagonists if they stimulated less than 5% of a full agonist response (data from human β1 taken from .

Figure 4. Correlation plot of the log K_D values for the turkey β-adrenoceptors compared with the human β-adrenoceptors.

Correlation plot of the log K_D values for the ligands in Table 1 for the three turkey β-adrenoceptors vs the three human β-adrenoceptors. The human adrenoceptor data is taken from and . Ligands are labelled as full agonists if the stimulated more than 90% of the response at the human β1-adrenoceptor, as partial agonists if they stimulated 5–90% of the full response and as antagonists if they stimulated less than 5% of a full agonist response (data from human β1 taken from .

Figure 5. CRE-SPAP production in response to cimaterol in the turkey β-adrenoceptors.

CRE-SPAP production in response to cimaterol in a) CHO tβtrunc cells, b) CHO tβ3C cells and c) CHO tβ4C cells in the absence and presence of propranolol. Bars represent basal CRE-SPAP production, that in response to 10 µM isoprenaline and that in response to 10 nM or 30 nM, 100 nM or 300 nM, or 1000 nM or 3000 nM propranolol alone. Data points are mean ± s.e.mean of triplicate determinations. These single experiments are representative of 3 separate experiments in each case. The Schild slopes in these experiments are a)1.10, b)1.15 and c)1.19.

Figure 6. CRE-SPAP production in response to CGP 12177 in the turkey β-adrenoceptors.

CRE-SPAP production in response to CGP12177 in a) CHO tβtrunc cells, b) CHO tβ3C cells and c) CHO tβ4C cells in the absence and presence of propranolol. Bars represent basal CRE-SPAP production, that in response to 10 µM isoprenaline and that in response to propranolol (range 10 nM to 100 µM) alone. Data points are mean ± s.e.mean of triplicate determinations. These single experiments are representative of 3 separate experiments in each case.

Figure 7. CRE-SPAP production in response to cimaterol in the turkey β-adrenoceptors.

CRE-SPAP production in response to cimaterol in a) CHO tβtrunc cells, b) CHO tβ3C cells and c) CHO tβ4C cells in the absence and presence of CGP12177. Bars represent basal CRE-SPAP production, that in response to 10 µM isoprenaline and that in response to 1 nM, 10 nM, 100 nM or 1000 nM CGP12177 alone. Data points are mean ± s.e.mean of triplicate determinations. These single experiments are representative of a) 5, b) 4 and c) 5 separate experiments.

Figure 8. ³H-cAMP accumulation in response to cyanopindolol in the turkey β-adrenoceptors.

³H-cAMP accumulation in response to cyanopindolol in a) CHO tβtrunc cells, b) CHO tβ3C cells and c) CHO tβ4C cells. Bars represent basal ³H-cAMP accumulation and that in response to 10 µM isoprenaline. Data points are mean ± s.e.mean of triplicate determinations. These single experiments are representative of a) 4, b) 5 and c) 5 separate experiments.

Figure 9. ³H-cAMP accumulation in response to cimaterol and CGP 12177 in the turkey β-adrenoceptors.

³H-cAMP accumulation in response to CGP12177 in a) CHO tβtrunc cells, b) CHO tβ3C cells and c) CHO tβ4C cells in the absence and presence of a fixed concentration of cimaterol. Bars represent basal ³H-cAMP accumulation, that in response to 10 µM isoprenaline and that in response to 0.3 nM, 1 nM, 3 nM or 10 nM cimaterol alone. Data points are mean ± s.e.mean of triplicate determinations. These single experiments are representative of a) 4, b) 5 and c) 4 separate experiments.