Constitutive precoupling to Gi and increased agonist potency in the α2B-adrenoceptor
Section snippets
Materials and methods
Mutagenesis ofα2B-AR. The cDNA of the human α2B-AR (a gift from Dr. R.J. Lefkowitz, Duke University [20]) was modified using polymerase chain reaction-mediated mutagenesis with Pfu DNA polymerase (Stratagene Cloning Systems, La Jolla, CA). The mutated DNA fragment was subcloned into a pMAM (Clontech Laboratories, Palo Alto, CA) expression vector containing the wild type α2B-AR cDNA [19], [21] and sequenced by automated DNA sequencing (Abi Prism).
Cell culture and transfection. Adherent CHO cells
Ligand binding properties and effects of PTX on agonist binding
Saturation binding experiments with [3H]RX821002 identified similar expression levels of wild-type α2B-AR (500 ± 220 (SD), n=6) and α2B-D109A receptors (580 ± 210 fmol/mg protein, n=14) in transfected CHO cells. Wild-type α2B-AR and α2B-D109A receptors also showed similar affinities for [3H]RX821002 (Kd=5.1±1.0 and Kd=4.5±1.1 nM, respectively).
The apparent binding affinities of most of the investigated α2-AR agonists were increased at the α2B-D109A compared to the wild-type α2B-AR (Table 1). The
Discussion
We investigated agonist binding affinities and cytosolic Ca2+ signal alterations in wild-type α2B-AR and α2B-D109A, stably expressed in CHO cells. The mutated receptor was shown to induce receptor precoupling to Gi, to have increased agonist affinity, to associate with increased [Ca2+]i in the absence of agonists, and to have enhanced potency and efficacy for agonists to induce Gi-mediated release of intracellular Ca2+. The antagonist RX821002 was able to counteract the increase in basal [Ca2+]i
Conclusions
Our findings indicate that the D3.49 aspartate residue in the conserved DRY sequence plays a critical role in controlling coupled and uncoupled conformations of α2B-AR. The D109A substitution causes constitutive signalling through PTX-sensitive G proteins and increases agonist potency and efficacy. The antagonist RX821002, which functioned as an inverse agonist at the mutated receptor, suppresses this spontaneous activation.
Acknowledgements
The authors thank Prof. Peter J. Slotte at the Department of Biochemistry and Pharmacy, Åbo Akademi University, for permission to use the Hitachi fluorometer. We thank Ms. Ulla Uoti and Ms. Anna-Mari Pekuri for technical help, Dr. Tuire Olli-Lähdesmäki for assistance in cell transfection, and M.Sc. Birgitta Sjöholm and M.Sc. Katariina Pohjanoksa for help in analysing the binding data. This study was supported by the Academy of Finland and by the Technology Development Centre of Finland.
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