Evidence for a second agonist conformation of the human β3-adrenoceptor

Jillian G Baker. Institute of Cell Signalling, University of Nottingham, Queen’s Medical Centre, Nottingham, NG7 2UH. UK.

The human β1-adrenoceptor (AR) is now generally considered to exist in at least 2 active sites or conformations (Konkar et al., 2000). Agonist acting thought the catecholamine site are readily inhibited by antagonists whereas agonist response occurring via the secondary site are relatively resistant to antagonism. Furthermore, some drugs appear to have agonist action at both sites (Baker et al., 2003). It appears that some agonist responses occurring via the human β3-AR are more sensitive to inhibition than others (Baker, this meeting, P010). The aim of this study was to determine whether there are different conformations of the human β3-AR.

CHO cells stably expressing the human β3-AR and a CRE-luciferase reporter gene were used. Measurements of 3H-cAMP accumulation and CRE-luciferase production were made as previously described (Baker et al., 2003).

ZD 7114 stimulated CRE-luciferase production that was best described by a 2-component response (log EC501 = -7.30±0.03, 68.5±1.6% of response, log EC502 = -4.78±0.06; n=18). ICI 118551 antagonised the first component to yield a log KD value of -6.03±0.04 (Fig 1), compatible with those previously described (Baker this meeting). A similar 2-component response was seen at the level of 3H-cAMP accumulation (log EC501 = -7.49±0.09, 61.6±3.1% of response, log EC502 = -5.41±0.25; n=5; log K D ICI 118551 for first component = -6.15±0.09, n=4). Fenoterol was a full agonist stimulating an increase in CRE-luciferase production that was 108.4±1.6% of the maximal isoprenaline response (log EC50 = -7.55±0.03, n=11). This was antagonised by ICI 118551 to yield a log KD value of-6.13±0.03 (n=18). Alprenolol stimulated an increase in CRE-luciferase production (log EC50 = -5.42±0.11; 54.1±5.5% of the isoprenaline) but this response was more resistant to antagonism by ICI 118551 (log KD value for ICI 118551 of -5.14±0.03; n=6). To look for further evidence of 2 agonist sites of the β3-AR alprenolol was incubated in the presence of fixed concentrations of fenoterol. Increasing concentrations of alprenolol reduced the fenoterol-stimulated CRE-luciferase production however, the inhibition occurred at lower concentrations of alprenolol than expected if both ligands were competing at the same site on the receptor (Fig 2; n=4).

Figure 1

Figure 2

Taken together, these data provide some evidence for the existence of a second conformation of the human β3-adrenoceptor akin to that of the β1-adrenoceptor.

Baker J.G. et al., (2003a). Mol. Pharmacol. 64, 679-688.
Baker J.G. et al., (2003b). Mol. Pharmacol. 63, 1312-1321.
Konkar A.A. et al., (2000) J. Pharmacol. Exp. Ther. 294, 923-932.