Mapping the Binding-Site Crevice of the D2 Receptor

Abstract

The binding sites of dopamine receptors are formed among their seven, mostly hydrophobic, membrane spanning segments and are accessible to charged, water-soluble agonists, like dopamine. Thus, each of these binding sites is contained within a water accessible crevice, the binding-site crevice, extending from the extracellular surface of the receptor into the plane of the membrane. The surface of this crevice is formed by residues that contact specific agonists and/or antagonists and other residues that may affect binding indirectly. To identify the residues that form the surface of the binding-site crevice in the human D2 receptor, the substituted-cysteine accessibility method (SCAM) is used. Consecutive residues in the membrane-spanning segments are mutated to cysteine, one at a time, and the mutant receptors are expressed in heterologous cells. If ligand binding to a cysteine-substitution mutant is near-normal, it is assumed that the structure of the mutant receptor, especially around the binding site, is similar to that of wild type and that the substituted cysteine lies in an orientation similar to that of the wild type residue. An additional advantage of this approach is that it can be determined whether a residue lines the binding-site crevice, even if mutation of the residue produces no functional change in the properties of the receptor.