The binding-site crevice of the D-4 dopamine receptor is coupled to three distinct sites of allosteric modulation

Most biogenic amine G protein-coupled receptors contain a conserved asparti c acid residue positioned near the intracellular side of the second transme mbrane-spanning (TMS) domain that is the primary site of allosteric modulat ion by sodium ions and pH. Recently, zinc ions and amiloride derivatives we re found to allosterically modulate antagonist binding to dopamine receptor s. In the current study, the wild-type D-4 dopamine receptor showed an 8-fo ld decrease in zinc affinity in the presence of 120 mM NaCl, but the bindin g of zinc to the neutral TMS2 D-4-D77N mutant was completely sodium-insensi tive. In contrast to zinc, methylisobutylamiloride (MIA) binding to the wil d-type D-4 receptor was virtually unaffected by sodium. In addition, the bi nding affinity for MIA was essentially unchanged in the presence of an IC50 concentration of zinc and vice versa. Furthermore, MIA binding affinity wa s decreased 4-fold for the D-4-D77N mutant and increased 30-fold for the TM S3 mutant D-4-M107V, even though the binding affinity for zinc was similar to the wild-type D-4 background for both mutants. These findings demonstrat e for the first time the existence of three distinct sites of allosteric mo dulation within a G protein-coupled receptor.