Role Of Homodimerisation In The Allosteric Interactions Between The Two β1-adrenoceptor Sites

K Gherbi, J Baker, S Briddon, S Hill. University of Nottingham, Nottingham, UK

We have previously reported allosteric interactions between the primary high affinity catecholamine and the secondary low affinity “CGP 12177” sites of the β1-adrenoceptor (1), although the nature of this secondary site is not yet fully understood. However, the transmembrane region (TM) 4 of the β1-adrenoceptor has recently been identified as essential for the observation of this second site pharmacology using cells expressing β1-adrenoceptors in which TM4 was swapped with that of the β2-adrenoceptor (2). The β1-adrenoceptor TM4 has also been reported to facilitate β1-adrenoceptor oligomerisation (3), suggesting a potential role of dimerisation in the secondary site CGP 12177 pharmacology. Here, we investigated whether the secondary site pharmacology observed at the β1-adrenoceptor is a consequence of allosteric interactions mediated across a homodimer interface.

This study used CHO-β1TM4 cells (expressing β1-adrenoceptors containing the β2-adrenoceptor TM4), and CHO-K1 cells transiently transfected with wild-type β1-adrenoceptors tagged with the N-terminal fragment of YFP (β1YFPN) and either a wild-type or non-ligand binding D138A β1-adrenoceptor mutant (4) tagged with the C-terminal fragment of YFP (β1YFPC, β1D138AYFPC, respectively). Irreversible bimolecular fluorescence complementation (BiFC) between the YFP fragments generated constrained β1-adrenoceptor dimers that were identified by the complemented YFP fluorescence. The method of infinite dilution on a temperature-controlled perfusion system in conjunction with the Zeiss LSM 710 confocal microscope (5) was then used to determine dissociation kinetics of the fluorescent ligand 3 nM BODIPY-TMR-CGP (6) in the absence and presence of 1 µM propranolol or 1 µM CGP12177. Cells were imaged every 2 s at 37 °C. 10 cells per each experiment (n) were analysed to measure fluorescence intensity changes over time. Using GraphPad Prism 5, dissociation data were fitted to a mono exponential decay equation and data are mean ± s.e.m. Statistical analysis was performed using one-way ANOVA.

The dissociation rate of 3 nM BODIPY-TMR-CGP (0.019 ± 0.006 min-1; n=5) in the absence of competitor ligands at wild-type β1-adrenoceptor BiFC homodimers was enhanced circa 10-fold in the presence of 1 µM CGP 12177 (0.186 ± 0.008 min-1; n=6) and 1 µM propranolol (0.189 ± 0.007 min-1; n=6). The magnitude of this effect was reduced to circa 3-fold in wild-type/D138A β1-adrenoceptor BiFC homodimers, with BODIPY-TMR-CGP dissociation rates of 0.054 ± 0.011 min-1 (n=5) in the absence of and 0.169 ± 0.010 min-1 (n=6) and 0.144 ± 0.009 min-1 (n=5) in the presence of 1 µM CGP 12177 and 1 µM propranolol, respectively. No allosteric effects of 1 µM CGP 12177 and 1 µM propranolol were observed in cells expressing β1-adrenoceptors containing β2-adrenoceptor TM4 swaps.

In conclusion, these data highlight a vital role of β1-adrenoceptor TM4 in the allosteric interactions between the two β1-adrenoceptor sites, and further suggest that the two β1-adrenoceptor sites are facilitated by β1-adrenoceptors in a homodimer formation.

This work was funded by the Medical Research Council.

(1) Salchow K et al. (2012). Proceedings of the British Pharmacological Society, http://www.pA2online.org/abstracts/Vol10Issue4abst170P.pdf.

(2) Baker JG et al. (2014). Mol Pharmacol 85: 811-829.

(3) Huang J et al. (2013). Nat Struct Mol Biol 20: 419-425.

(4) Baker JG et al. (2008). Mol Pharmacol 74: 1246-1260.

(5) May LT et al. (2010). Mol Pharmacol 78: 511-523.

(6) Gherbi K et al. (2014). Br J Pharmacol In press DOI: 10.1111/bph.12858.