A “locked-on,” constitutively active mutant of the adenosine A1 receptor

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Abstract

We studied the wild-type human adenosine A1 receptor and three mutant receptors, in which the glycine at position 14 had been changed into an alanine, a leucine, or a threonine residue. All receptors were characterized in radioligand binding experiments, the wild-type and the Gly14Thr mutant receptor in greater detail. Both receptors were allosterically modulated by sodium ions and PD81,723 (2-amino-4,5-dimethyl-3-thienyl-[3(trifluoromethyl)-phenyl]methanone), although in a different way. All mutant receptors appeared to be spontaneously or “constitutively” active in a [35S]GTPγS binding assay, the first demonstration of the existence of such CAM (constitutively active mutant) receptors for the adenosine A1 receptor. The Gly14Thr mutant receptor was also constitutively active in another functional assay, i.e., the inhibition of forskolin-induced cAMP production in intact cells. Importantly, this mutant displayed a peculiar “locked-on” phenotype, i.e., neither agonist nor inverse agonist was capable of modulating the basal activity in both the GTPγS and the cAMP assay, unlike the wild-type and the two other mutant receptors.

Introduction

G protein-coupled receptors (GPCRs) may occur in at least two conformational states, one inactive (R), the other active (R*) (Leff, 1995). To study these different receptor states, constitutively active mutant (CAM) receptors have been engineered as a research tool. CAM receptors are thought to have an increased proportion of their population in the R* conformation, i.e., they mimic the active state of the receptor. Consequently, they show a higher level of spontaneous receptor activity, even in the absence of an agonist (De Ligt et al., 2000, Leurs et al., 1998, Milligan et al., 1997, Parnot et al., 2002). However, a CAM adenosine A1 receptor has not been reported yet.

Other strategies to discriminate between the various receptor states include the use of appropriate modulators of ligand binding. Examples of such modulators are GTP, sodium ions, and, specific for the adenosine A1 receptor, PD81,723 (2-amino-4,5-dimethyl-3-thienyl-[3(trifluoromethyl)-phenyl]methanone), which is an allosteric enhancer of agonist binding. All three modulators influence ligand binding to the adenosine A1 receptor by intervening with the existing receptor equilibrium in a different manner. First, GTP uncouples the G protein from the receptor, and shifts the receptor equilibrium to the R conformation. This results in a decreased affinity of agonists. Secondly, sodium ions also shift the equilibrium to the inactive receptor state (R). They probably exhibit their effect by interacting with a highly conserved aspartate residue in the second transmembrane helix of virtually all GPCRs, Asp55 in the human adenosine A1 receptor (Barbhaiya et al., 1996). Finally, PD81,723 enhances adenosine A1 agonist binding (Bruns and Fergus, 1990). It presumably acts by increasing the number of adenosine A1 receptors in the R* conformation, and so it increases the affinity of adenosine receptor agonists. The shifts in ligand binding properties induced by these three modulators can also be used to discriminate between the different classes of ligands, i.e., agonists, inverse agonists and neutral antagonists. For instance, GTP has been used to discriminate between full and partial agonists (Van der Wenden et al., 1995) or neutral antagonists and inverse agonists (Van Calenbergh et al., 2002).

In the present study we focussed on the wild-type human adenosine A1 receptor and a mutant receptor previously described by us (Rivkees et al., 1999). In this receptor the glycine residue at position 14 had been changed into a threonine (Gly14Thr A1). Adenosine A1 receptor agonists, e.g., N6-cyclopentyladenosine (CPA), displayed a higher affinity for this Gly14Thr A1 mutant than for the wild-type adenosine A1 receptor. In retrospect we reasoned this is an indication that the Gly14Thr A1 receptor might be spontaneously active and behave as a CAM receptor. For comparison, we also introduced two new mutations at this position, an alanine (Gly14Ala) or a leucine (Gly14Leu) residue.

Various methods were used to establish the pharmacological effects of the Gly14-mutations. Saturation analyses revealed differences in R:R* ratios for the Gly14Thr A1 receptor compared to the wild-type adenosine A1 receptor. Allosteric modulation of ligand binding provided further insights into this conformational equilibrium. Finally, receptor activation and functionality of the various (mutant) adenosine A1 receptors was measured with [35S]GTPγS binding studies and cAMP determinations. We discovered that all three mutants display constitutive activity and that the Gly14Thr mutant receptor possesses a very peculiar “locked-on” phenotype.

Section snippets

Chemicals

[35S]GTPγS (1250 Ci/mmol), [3H]DPCPX (1,3-dipropyl,8-cyclopentylxanthine, 112 Ci/mmol), [3H]CCPA (2-chloro,N6-cyclopentyladenosine, 55 Ci/mmol) and [3H]cAMP (25 Ci/mmol) were obtained from Perkin Elmer Life Sciences (Dreieich, Germany). Adenosine deaminase (ADA), DEAE dextran, chloroquine, and dithiothreitol were purchased from Sigma. EDTA, MgCl2, GDP, and GTPγS were obtained from Boehringer (Mannheim, Germany). CPA, DPCPX and 8-cyclopentyltheophylline (CPT) were purchased from Research

Results

As a start we determined IC50 values of CPA and DPCPX for the wild-type and three Gly14-mutated receptors (Table 1). Compared to the wild-type adenosine A1 receptor, the IC50 value of CPA was similar for the Gly14Ala and Gly14Leu A1 receptor. Interestingly, the IC50 value of CPA for the Gly14Thr A1 receptor was significantly lower than for the three other constructs, namely 5.04 nM versus 436 (wild-type), 459 (Gly14Ala), and 551 (Gly14Leu) nM, respectively. On the other hand, DPCPX had only

Discussion

Mutation of the glycine residue at position 14 (Gly14) of the human adenosine A1 receptor into a threonine (as present in the human adenosine A2A receptor) had been shown to increase agonist affinity (Rivkees et al., 1999). Since this finding might be indicative for the receptor's constitutive activity, we now analysed the Gly14Thr A1 receptor in more detail. For comparison, two other mutations at this position, Gly14Ala and Gly14Leu were included in this study. The rationale for this choice

Conclusions

In conclusion, in this study we described, for the first time, three CAM (Gly14Ala, Gly14Leu, and Gly14Thr) adenosine A1 receptors, as found in [35S]GTPγS binding experiments. Moreover, it seemed that each mutant A1 receptor had distinctive characteristics. Although the Gly14Thr A1 receptor was able to bind ligands, such as CPA and DPCPX (Table 1, Table 2, Table 3), it was not susceptible to further receptor (de)activation, as measured with [35S]GTPγS binding (Fig. 2) or cAMP production (Table 4

Acknowledgements

The authors (R.d.L., R.L., and A.P.IJ.) acknowledge financial support from the EU BIOMED2 programme “Inverse agonism. Implications for drug design” (#BMH4-CT97-2152).

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