Selective reconstitution of human D4 dopamine receptor variants with G(i)alpha subtypes

G protein-coupled receptors (GPCRs) are seven-transmembrane (TM) helical pr oteins that bind extracellular molecules and transduce signals by coupling to heterotrimeric G proteins in the cytoplasm. The human D4 dopamine recept or is a particularly interesting GPCR because the polypeptide loop linking TM helices 5 and 6 (loop i3) may contain from 2 to 10 similar direct hexade capeptide repeats. The precise role of loop i3 in D4 receptor function is n ot known. To clarify the role of loop i3 in G protein coupling, we construc ted synthetic genes for the three main Df receptor variants. D4-2, D-4-4, a nd D4-7 receptors contain 2, 4, and 7 imperfect hexadecapeptide repeats in loop i3, respectively. We expressed and characterized the synthetic genes a nd found no significant effect of the D4 receptor polymorphisms on antagoni st or agonist binding. We developed a cell-based assay where activated D4 r eceptors coupled to a Pertussis toxin-sensitive pathway to increase intrace llular calcium concentration. Studies using receptor mutants showed that th e regions of loop i3 near TM helices 5 and 6 were required for G protein co upling. The hexadecapeptide repeals were not required for G protein-mediate d calcium flux. Cell membranes containing expressed D4 receptors and recept or mutants were reconstituted with purified recombinant G protein alpha sub units. The results show that each D4 receptor variant is capable of couplin g to several G(i)alpha subtypes. Furthermore, there is no evidence of any q uantitative difference in G protein coupling related to the number of hexad ecapeptide repeats in loop i3. Thus? loop i3 is required for D4 receptors t o activate G proteins. However, the polymorphic region of the loop does not appear to affect the specificity or efficiency of G(i)alpha coupling.