Aim: To define roles of the third intracellular loop (IL3) length of G-protein coupled receptors in conferring the specificity for receptor binding and G-protein coupling.
Methods: By polymerase chain reaction (PCR), the IL3 of D2 receptor was replaced with the counter part of AT1 receptor which has the shortest loop among all G-protein coupled receptors. D2/AT1 receptor cDNA was then stably transfected into Chinese hamster ovary cells and a clone with high level expression was obtained for receptor binding and agonist-induced phosphatidylinositols (PI) turnover experiments.
Results: Comparing to the D2 receptor, D2/AT1 chimeric receptor had lower affinities for all D2 receptor antagonists tested (spiperone, haloperidol, (+)-butaclamol, chlopromazine, clozapine, trifluoperdazine) and different affinity profiles to agonists (apomorphine, dopamine, quinpirole, bromocriptine). But the chimeric receptor failed to couple to G-protein and subsequent stimulation of PI turnover.
Conclusion: The length of IL3 of D2 receptor participates defining recpetor binding sites conformation, and structure beyond IL3 may affect receptor G-protein coupling.