The fourth transmembrane segment of the dopamine D2 receptor: Accessibility in the binding-site crevice and position in the transmembrane bundle

Citation
Ja. Javitch et al., The fourth transmembrane segment of the dopamine D2 receptor: Accessibility in the binding-site crevice and position in the transmembrane bundle, BIOCHEM, 39(40), 2000, pp. 12190-12199
Citations number
51
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMISTRY
ISSN journal
00062960 → ACNP
Volume
39
Issue
40
Year of publication
2000
Pages
12190 - 12199
Database
ISI
SICI code
0006-2960(20001010)39:40<12190:TFTSOT>2.0.ZU;2-7
Abstract
The binding site of the dopamine D2 receptor, like that of homologous G-pro tein-coupled receptors (GPCRs), is contained within a water-accessible crev ice formed among its seven transmembrane segments (TMSs). Using the substit uted-cysteine-accessibility method (SCAM), we are mapping the residues that contribute to the surface of this binding-site crevice. We have mutated to cysteine, one at a time, 21 consecutive 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 which cysteine was substituted for residues in the cytoplasmic half of TM4, treatment with the reagents had no effect on binding, consistent with these residues being inaccessible and with the low-resolution structure of the homologous rhodopsin, in which TM3 and TM5 occlude the cytoplasmic half of TM4. Although hydrophobicity analysis positions the C-terminus of TM4 a t 4.64, Pro-Pro and Pro-X-Pro motifs, which are known to disrupt ct-helices , occur at position 4.59 in a number of homologous GPCRs, The SCAM data wer e consistent with a C-terminus at 4.58, but it is also possible that the ct -helix extends one additional turn to 4.62 in the D2 receptor, which has a single Pro at 4.59. In homologous GPCRs, the high degree of sequence variat ion between 4.59 and 4.68 is more characteristic of a loop domain than a he lical segment. This region is shown here to be very conserved within functi onally related receptors, suggesting an important functional role for this putative nonhelical domain. This inference is supported by observed ligand- specific effects of mutations in this region and by the predicted spatial p roximity of this segment to known ligand binding sites in other TMs.