title: The binding-site crevice of the D4 dopamine receptor is coupled to three distinct sites of allosteric modulation.
abstract: Most biogenic amine G protein-coupled receptors contain a conserved aspartic acid residue positioned near the intracellular side of the second transmembrane-spanning (TMS) domain that is the primary site of allosteric modulation by sodium ions and pH. Recently, zinc ions and amiloride derivatives were found to allosterically modulate antagonist binding to dopamine receptors. In the current study, the wild-type D4 dopamine receptor showed an 8-fold decrease in zinc affinity in the presence of 120 mM NaCl, but the binding of zinc to the neutral TMS2 D4-D77N mutant was completely sodium-insensitive. In contrast to zinc, methylisobutylamiloride (MIA) binding to the wild-type D4 receptor was virtually unaffected by sodium. In addition, the binding affinity for MIA was essentially unchanged in the presence of an IC(50) concentration of zinc and vice versa. Furthermore, MIA binding affinity was decreased 4-fold for the D4-D77N mutant and increased 30-fold for the TMS3 mutant D4-M107V, even though the binding affinity for zinc was similar to the wild-type D4 background for both mutants. These findings demonstrate for the first time the existence of three distinct sites of allosteric modulation within a G protein-coupled receptor.
authors.affiliation: Department of Pharmacology, University of Mississippi, University, Mississippi 38677-1848, USA. john@olemiss.edu
authors.forename: J A
authors.initials: JA
authors.lastname: Schetz
authors.forename: D R
authors.initials: DR
authors.lastname: Sibley
mesh_terms: D000584:Amiloride / Q000031:analogs & derivatives / Q000737:chemistry; D001665:Binding Sites / Q000187:drug effects / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000276:immunology; D019204:GTP-Binding Proteins / Q000378:metabolism; D007202:Indicators and Reagents; D008566:Membranes / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D011487:Protein Conformation; D011869:Radioligand Assay; D017448:Receptors, Dopamine D2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D050638:Receptors, Dopamine D4; D012964:Sodium / Q000494:pharmacology; D014162:Transfection; D015032:Zinc / Q000378:metabolism
chemical_list: D007202:Indicators and Reagents; D017448:Receptors, Dopamine D2; D050638:Receptors, Dopamine D4; D000584:Amiloride; D004247:DNA; C056369:5-(N-methyl-N-isobutyl)amiloride; D012964:Sodium; D019204:GTP-Binding Proteins; D015032:Zinc