A pharmacological characterisation of the thermostabilized turkey β-adrenoceptor (tβtrunc-M23)

Jillian Baker1, Christopher Tate2

1University of Nottingham, Nottingham, UK, 2MRC Laboratory of Molecular Medicine, Cambridge, UK

The turkey β-adrenoceptor (AR) has 82% sequence homology with the human β1-AR and its structure was recently determined to 2.7 Å resolution (Warne et al., 2008). This receptor has deletions in the N and C termini and, in order to obtain crystals that diffracted to a high resolution contains, 6 point mutations were required to thermostabilse the receptor (R67S, M90V, Y227A, A282L, F327A, F338M; Warne et al., 2008). This study examined the pharmacological characteristics of this receptor (tβtrunc-M23).

Turkey βtrunc-M23 DNA was transfected into CHO-K1 cells stably expressing a CRE-SPAP reporter gene. Initial binding studies were performed on transient mixed populations of cells. A stable cell line was also made to undertake a detailed pharmacological analysis. 3H-CGP 12177 whole cell binding, CRE-gene transcription and 3H-cAMP accumulation assays were performed as previously described (Baker 2005). The binding characteristics were very similar whether determined from transiently transfected mixed populations or from the stable cell line. The stable cell line data is reported here.

tβtrunc-M23 bound some β-AR ligands with high affinity (yielding log KD values of: propranolol -7.63±0.04, n=5, timolol -7.70±0.03, n=5, carazolol -8.83±0.06, n=7 and cyanopindolol -9.77±0.02, n=6). Other ligands had lower affinity (e.g. log KD CGP 20712A -6.38±0.11, n=7; ICI 118551 -6.49±0.03, n=7). Agonist responses was seen to many AR agonists (e.g. isoprenaline (isop) log EC50 -7.36±0.12, n=17; cimaterol -7.31±0.06, 96.6% isop max, n=16; formoterol -7.75±0.08, 106.7±2.3% isop max, n=14;). These responses were antagonised by propranolol in a competitive manner to yield log KD values of -7.63±0.10, n-13; -8.28±0.05, n=10 and -8.24±0.02, n=13 respectively and Schild slopes of 1. CGP 12177 stimulated a partial agonist response (log EC50 = -6.78±0.06, 21.8±2.2% isop max response, n=10) however this required higher concentrations of ligands to antagonise the response (log KD values for propranolol and ICI 118551 of -6.12±0.08 and -5.51±0.12, n=5 respectively). CGP 21177 however inhibited other agonist responses with high affinity (e.g log KD -9.04±0.09, n=9; -9.08±0.15, n=6 in the presence of cimaterol and formoterol respectively). Thus the affinity of CGP 12177 as determined from 3H-CGP 12177 binding and antagonism of other agonists was significantly higher than, and therefore at odds with, the EC50 value required to stimulate an agonist response. Cyanopindolol stimulated a small cAMP response (8.2±1.2% isop max), however this was best described by a two-component fit (log EC501 -9.34±0.07, EC502 -7.09±0.05, EC501 = 40.8±0.5% total response, n=5).

In conclusion, the turkey βtrunc-M23 receptor is a functionally active β-AR which appears to exist in at least two active agonist conformations in a similar manner to that of mammalian β1-ARs (high affinity catecholamine conformation, low affinity CGP 12177 conformation; Konkar et al. 2000; Baker 2005).

JGB is a Wellcome Trust Clinician Scientist

Baker JG (2005) J Pharmacol Exp Ther 313: 1163-1171

Konkar AA et al., (2000) J Pharmacol Exp Ther 294: 926-932

Warne T et al., (2008) Nature 454: 486-491.