A CYSTEINE RESIDUE IN THE 3RD MEMBRANE-SPANNING SEGMENT OF THE HUMAN D-2-DOPAMINE RECEPTOR IS EXPOSED IN THE BINDING-SITE CREVICE

The binding site in G-protein-Linked neurotransmitter receptors is for med among their membrane-spanning segments. Because the binding site i s in the plane of the bilayer and is accessible to charged, water-solu ble agonists, it must lie in a crevice open to the extracellular, aque ous medium. Information about the structure of these receptors can be obtained by identifying the residues in the membrane-spanning segments which face this water-filled crevice. Human D-2 dopamine receptor was expressed in human embryonic kidney 293 cells. Small, charged, sulfhy dryl-specific methanethiosulfonate (MTS) derivatives irreversibly inhi bited the binding of the D-2-specific antagonist [H-3]YM-09151-2 to th ese cells. The highly polar MTS derivatives should react with cysteine sulfhydryl groups only at the water-accessible surface of the recepto r, which includes the surface of the binding-site crevice. In contrast , these reagents will have little access to sulfhydryls facing the lip id bilayer or buried in the protein interior. Positively charged MTS r eagents irreversibly inhibited binding several hundredfold faster than a negatively charged MTS reagent, consistent with the affinity of-the binding site for positively charged dopamine agonists and antagonists . Furthermore, both agonists and antagonists of the D-2 receptor prote cted against irreversible inhibition by the MTS reagents. To identify the susceptible cysteine, we mutated, one at a time, five transmembran e and two extracellular cysteine residues to serine. Only the mutation of Cys(118) to serine decreased the susceptibility of antagonist bind ing to irreversible inhibition by the MTS reagents. Thus, Cys(118), a residue in the middle of the third membrane-spanning segment, is expos ed in the D-2 receptor binding-site crevice.