Allosteric modulation and constitutive activity of fusion proteins between the adenosine A1 receptor and different 351Cys-mutated Gi α-subunits

We studied fusion proteins between the human adenosine A(1) receptor and different (351)Cys-mutated G(il) alpha-subunits (A(1)-G(i)alpha) with respect to two important concepts in receptor pharmacology, i.e. allosteric modulation and constitutive activity/inverse agonism. The aim of our study was twofold. We first analysed whether such fusion products are still subject to allosteric modulation, and, secondly, we investigated the potential utility of the fusion proteins to study constitutive receptor activity. We determined the pharmacological profile of nine different A(1)-G(i)alpha fusion proteins in radioligand binding studies. In addition, we performed [S-35]GTPgammaS binding experiments to study receptor and G protein activation of selected A(1)-G(i)alpha fusion proteins. Compared to unfused adenosine A, receptors, the affinity of N-6-cyclopentyladenosine (CPA) at wild-type A(1)-G(i)alpha fusion proteins ((351)Cys) increased more than eightfold, while the affinity of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) did not change significantly. Furthermore, we showed that the allosteric enhancer of agonist binding, PD81,723 (2-amino-4,5-dimethyl-3-thienyl-[3-(trifluoromethyl)-phenyl]methanone), elicited similar effects on ligand binding; i.e. CPA binding to the A(1)-G(i)alpha fusion proteins was enhanced, whereas the affinity of DPCPX was hardly affected. Moreover, sodium ions were unable to decrease agonist binding to the majority of the A(1)-G(i)alpha fusion proteins, presumably because they exhibit their effect through uncoupling of the R-G complex. From [S-35]GTPgammaS binding experiments, we learned that all the A(1)-G(i)alpha fusion proteins tested had a higher basal receptor activity than the unfused adenosine A, receptor, thereby providing improved conditions to observe inverse agonism. Moreover, the maximal CPA-induced stimulation of basal [S-35]GTPgammaS binding was increased for the five A(1)-G(i)alpha fusion proteins tested, whereas the inhibition induced by 8-cyclopentyltheophylline (CPT) was more pronounced at (351)Cys, (351)Ile, and (351)Val A(1)-G(i)alpha fusion proteins. Thus, the maximal receptor (de)activation depended on the amino acid at position 351 of the G(i) alpha-subunit. In conclusion, A(1)-G(i)alpha fusion proteins, especially with (351)Cys and (351)Ile. can be used as research tools to investigate inverse agonism, due to their increased readout window in [S-35]GTPgammaS binding experiments. (C) 2004 Elsevier B.V. All rights reserved.