Investigation into the pharmacology of the adenosine A2A receptor stabilised in an active-state conformation

Kirstie Bennett1, Guillaume Lebon2, Ben Tehan1, Christopher Tate2, Christopher Langmead1. 1Heptares Therapeutics, Welwyn Garden City, Herts., UK, 2MRC Laboratory of Molecular Biology, Cambridge, UK.

StaR® technology works by introducing a series of point mutations into G protein coupled receptors (GPCRs) to increase their thermostability towards the conformation of choice. StaR technology has been used to solve crystal structures of the adenosine A2A receptor in both active (agonist-bound) and inactive (inverse agonist-bound) states (Dore et al., 2011; Lebon et al, 2011a). Here we investigate the effect of stabilising the A2A receptor into the active state on G protein signalling using a family of progressively thermostable A2A StaRs.

To create active stabilised versions of the A2A receptor a series of point mutations were introduced into the receptor and receptor thermostability assessed (described in Lebon et al., 2011b). A series of A2A agonist StaRs were selected (GL0, GL23, GL26 and GL31) and the affinity of a panel of ligands assessed using [3H]NECA binding assays in CHO membranes transiently expressing receptor. To assess the impact of thermostabilisation on receptor function a CisBio cAMP assay was used to measure Gαs activation. T-REx CHO cell lines were generated placing receptor expression levels under control of a tetracycline operon allowing alteration in receptor number by titrating doxycycline levels. Cells were seeded onto half-area 96-well plates (25,000/well), receptor expression induced (0-10 ng/mL doxycycline; 16 h) before cells were challenged (30 min; 25 °C) with NECA diluted in assay buffer (Krebs pH 7.4 supplemented with 2U/mL adenosine deaminase and 100 uM Ro-201174) or assay buffer alone (basal). Assay was terminated and read as per manufacturer’s instructions.

For the native A2A receptor it was found that increasing doxycycline concentrations (0-1 mg/mL) yielded an increase in basal cAMP accumulation levels (up to 1.39-fold over basal; n = 3) consistent with previous reports that the A2A receptor is constitutively active. NECA was able to produce a response in this system in the absence of doxycycline (pEC50 ± S.E.M = 9.18 ± 0.08; n = 4), but not in the parental cell line, suggesting the cell line was leaky. As A2A receptor expression was increased so did the potency of NECA (i.e. at 1 ng/mL doxycycline pEC50 ± S.E.M = 10.24 ± 0.41). For each of the active state StaRs increasing doxycycline concentrations caused an increase in basal cAMP levels (GL0 3.23-fold, GL23 3.23-fold, GL26 3.72-fold, GL31 3.77-fold; n = 3) though the ability of NECA to further activate cAMP accumulation was greatly reduced (pEC50 ± S.E.M at 1 ng/mL doxycycline GL0 = 7.46 ± 0.39; GL23 = 8.18 ± 0.18, GL26 = 7.84 ± 0.36, GL31 = 8.17 ± 0.15; n = 2-5). The affinity of NECA for each StaR was not significantly different from native receptor (p>0.05; one-way ANOVA; KD ± S.E.M. WT = 12.36 ± 5.20; GL0 = 6.44 ± 1.31; GL23 = 4.12 ± 0.97; GL26 4.18 ± 1.29; GL31 = 2.82 ± 0.68).

The difference between native A2A and the simplest active state StaR (GL0) is mutation of leucine 48 (L2.46 in Ballesteros-Weinstein numbering) to alanine. Analysis of the A2A-NECA crystal structure (PDB code 2YDV) shows that it is in a conformation close to that expected for a fully-activated receptor, except that the cytoplasmic end of helix 6 has not moved outward as far as would be predicted from structures of b2-adrenoceptor. It appears from the A2A-NECA structure that the L2.46A mutation allows the movement of proline 285 (P7.50) within the NPXXY motif, thus stabilising the active conformation of TM7 and the G protein binding site in general. This suggests that L2.46 is in an important region of the receptor for G protein activation and that it plays a key role in stabilising intermediates in the activation of GPCRs.

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Lebon G, Warne T, Edwards PC, Bennett K, Langmead CJ, Leslie AG et al (2011a) Agonist-bound adenosine A2A receptor structures reveal common features of GPCR activation. Nature 18; 474 (7352)521-5.

Lebon G, Bennett K, Jazayeri A, Tate CG (2011b) Thermostabilisation of an agonist-bound conformation of the human adenosine A2A receptor. J Mol Biol 10; 409 (3) 298-310.