Selective reconstitution of human D4 dopamine receptor variants with Gi alpha subtypes.
G protein-coupled receptors (GPCRs) are seven-transmembrane (TM) helical proteins that bind extracellular molecules and transduce signals by coupling to heterotrimeric G proteins in the cytoplasm. The human D4 dopamine receptor 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 hexadecapeptide repeats. The precise role of loop i3 in D4 receptor function is not known. To clarify the role of loop i3 in G protein coupling, we constructed synthetic genes for the three main D4 receptor variants. D4-2, D4-4, and D4-7 receptors contain 2, 4, and 7 imperfect hexadecapeptide repeats in loop i3, respectively. We expressed and characterized the synthetic genes and found no significant effect of the D4 receptor polymorphisms on antagonist or agonist binding. We developed a cell-based assay where activated D4 receptors coupled to a Pertussis toxin-sensitive pathway to increase intracellular calcium concentration. Studies using receptor mutants showed that the regions of loop i3 near TM helices 5 and 6 were required for G protein coupling. The hexadecapeptide repeats were not required for G protein-mediated calcium flux. Cell membranes containing expressed D4 receptors and receptor mutants were reconstituted with purified recombinant G protein alpha subunits. The results show that each D4 receptor variant is capable of coupling to several G(i)alpha subtypes. Furthermore, there is no evidence of any quantitative difference in G protein coupling related to the number of hexadecapeptide repeats in loop i3. Thus, loop i3 is required for D4 receptors to activate G proteins. However, the polymorphic region of the loop does not appear to affect the specificity or efficiency of G(i)alpha coupling.
Kazmi
M A
MA
Howard Hughes Medical Institute, Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, New York, New York 10021, USA.
Snyder
L A
LA
Cypess
A M
AM
Graber
S G
SG
Sakmar
T P
TP
D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000037:antagonists & inhibitors / Q000378:metabolism; D002460:Cell Line; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005813:Genes, Synthetic; D014644:Genetic Variation; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis / Q000138:chemical synthesis / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary; D019257:Quinpirole / Q000494:pharmacology; D017448:Receptors, Dopamine D2 / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D050638:Receptors, Dopamine D4; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000138:chemical synthesis; D011994:Recombinant Proteins / Q000138:chemical synthesis / Q000378:metabolism; D013134:Spiperone / Q000378:metabolism
C493599:DRD4 protein, human; C493600:Drd4 protein, mouse; D010446:Peptide Fragments; D017448:Receptors, Dopamine D2; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D050638:Receptors, Dopamine D4; D019257:Quinpirole; D013134:Spiperone; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D002118:Calcium