| Abstract
| - The binding site of the dopamine D2 receptor, like that of homologous G-protein-coupledreceptors (GPCRs), is contained within a water-accessible crevice formed among its seven transmembranesegments (TMs). Using the substituted-cysteine-accessibility method (SCAM), we are mapping the residuesthat 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, whereasbound antagonist only protected N521.50C from reaction. Except for A381.36C, none of the substitutedcysteine 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 showedthat Pro1.48 bends the extracellular portion of TM1 inward toward TM2 and TM7. Analysis of theconversation of residues in the extracellular portion of TM1 of opsins showed a pattern consistentwith α-helical structure with a conserved face. In contrast, this region in catecholamine receptors ispoorly conserved, suggesting a lack of critical contacts. Thus, in catecholamine receptors in the absenceof Pro1.48, TM1 may be straighter and therefore further from the helix bundle, consistent with the apparentlack of conserved contact residues. When examined in the context of a model of the D2 receptor, theaccessible 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 stateof the receptor.
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