The Fourth Transmembrane Segment of the Dopamine D2 Receptor: Accessibility in the Binding-Site Crevice and Position in the Transmembrane Bundle
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- 作者：Jonathan A. Javitch ; Lei Shi ; Merrill M. Simpson ; Jiayun Chen ; Victor Chiappa ; Irache Visiers ; Harel Weinstein ; and Juan A. Ballesteros
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：October 10, 2000
- 年：2000
- 卷：39
- 期：40
- 页码：12190 - 12199
- 全文大小：365K
- 年卷期：v.39,no.40(October 10, 2000)
- ISSN：1520-4995
文摘
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 (TMSs). Using the substituted-cysteine-accessibility method (SCAM), we are mapping the residuesthat contribute to the surface of this binding-site crevice. We have mutated to cysteine, one at a time, 21consecutive residues in the fourth TMS (TM4). Eleven of these mutants reacted with charged sulfhydryl-specific reagents, and bound antagonist protected nine of these from reaction. For the mutants in whichcysteine was substituted for residues in the cytoplasmic half of TM4, treatment with the reagents had noeffect on binding, consistent with these residues being inaccessible and with the low-resolution structureof the homologous rhodopsin, in which TM3 and TM5 occlude the cytoplasmic half of TM4. Althoughhydrophobicity analysis positions the C-terminus of TM4 at 4.64, Pro-Pro and Pro-X-Pro motifs, whichare known to disrupt 
mages/gifchars/alpha.gif" BORDER=0>-helices, occur at position 4.59 in a number of homologous GPCRs. The SCAMdata were consistent with a C-terminus at 4.58, but it is also possible that the mages/gifchars/alpha.gif" BORDER=0>-helix extends one additionalturn to 4.62 in the D2 receptor, which has a single Pro at 4.59. In homologous GPCRs, the high degreeof sequence variation between 4.59 and 4.68 is more characteristic of a loop domain than a helical segment.This region is shown here to be very conserved within functionally related receptors, suggesting an importantfunctional role for this putative nonhelical domain. This inference is supported by observed ligand-specificeffects of mutations in this region and by the predicted spatial proximity of this segment to known ligandbinding sites in other TMs.