The first transmembrane segment of the dopamine D2 receptor: accessibility in the binding-site crevice and position in the transmembrane bundle
Shi, L.; Simpson, M.M.; Ballesteros, J.A.; Javitch, J.A.
Biochemistry 40(41): 12339-12348
ISSN/ISBN: 0006-2960 PMID: 11591153 DOI: 10.1021/bi011204a
The binding site of the dopamine D2 receptor, like that of homologous G-protein-coupled receptors (GPCRs), is contained within a water-accessible crevice formed among its seven transmembrane segments (TMs). Using the substituted-cysteine-accessibility method (SCAM), we are mapping the residues that contribute to the surface of this binding-site crevice. We have now mutated to cysteine, one at a time, 21 consecutive residues in TM1. Six of these mutants reacted with charged sulfhydryl reagents, whereas bound antagonist only protected N521.50C from reaction. Except for A381.36C, none of the substituted cysteine mutants in the extracellular half of TM1 appeared to be accessible. Pro1.48 is highly conserved in opsins, but absent in catecholamine receptors, and the high-resolution rhodopsin structure showed that Pro1.48 bends the extracellular portion of TM1 inward toward TM2 and TM7. Analysis of the conversation of residues in the extracellular portion of TM1 of opsins showed a pattern consistent with a-helical structure with a conserved face. In contrast, this region in catecholamine receptors is poorly conserved, suggesting a lack of critical contacts. Thus, in catecholamine receptors in the absence of Pro1.48, TM1 may be straighter and therefore further from the helix bundle, consistent with the apparent lack of conserved contact residues. When examined in the context of a model of the D2 receptor, the accessible residues in the cytoplasmic half of TM1 are at the interface with TM7 and with helix 8 (H8). We propose the existence of critical contacts of TM1, TM7, and H8 that may stabilize the inactive state of the receptor. Reprinted by permission of the publisher.