Ja. Javitch et al., A CYSTEINE RESIDUE IN THE 3RD MEMBRANE-SPANNING SEGMENT OF THE HUMAN D-2-DOPAMINE RECEPTOR IS EXPOSED IN THE BINDING-SITE CREVICE, Proceedings of the National Academy of Sciences of the United Statesof America, 91(22), 1994, pp. 10355-10359
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.