Section 10
Chapter 9,345

Residues in the seventh membrane-spanning segment of the dopamine D2 receptor accessible in the binding-site crevice

Fu, D.; Ballesteros, J.A.; Weinstein, H.; Chen, J.; Javitch, J.A.

Biochemistry 35(35): 11278-11285


ISSN/ISBN: 0006-2960
PMID: 8784181
DOI: 10.1021/bi960928x
Accession: 009344341

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The binding site of the dopamine D2 receptor, like that of other homologous G-protein-coupled receptors, is contained within a water-accessible crevice formed among its seven membrane-spanning segments. Using the substituted-cysteine accessibility method, we previously mapped the residues that form the surface of the binding-site crevice in the third and fifth membrane-spanning segments (M3 and M5). We have now mutated to cysteine, one at a time, 26 consecutive residues in and flanking the seventh membrane-spanning segment (M7) and expressed the mutant receptors in HEK 293 cells. Nine of these mutants reacted with charged, hydrophilic, lipophobic, sulfhydryl-specific reagents, added extracellularly, and were protected from reaction by a reversible dopamine antagonist, sulpiride. Thus, we infer that the side chains of these residues are in the water-accessible surface of the binding-site crevice. The pattern of accessibility of the cysteine-substitution mutants is consistent with M7 being a kinked alpha-helix.