Vol. 296, Issue 2, 359-363, February 2001

The Binding-Site Crevice of the D₄ Dopamine Receptor Is Coupled to Three Distinct Sites of Allosteric Modulation

John A. Schetz and David R. Sibley

Department of Pharmacology, University of Mississippi, University, Mississippi (J.A.S.); and Molecular Neuropharmacology Section, Experimental Therapeutics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland (D.R.S.)

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 IC₅₀ 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.