Conserved polar residues in the transmembrane domain of the human tachykinin NK2 receptor:: functional roles and structural implications

被引:49
作者
Donnelly, D [1 ]
Maudsley, S
Gent, JP
Moser, RN
Hurrell, CR
Findlay, JBC
机构
[1] Univ Leeds, Fac Biol Sci, Sch Biomed Sci, Leeds LS2 9JT, W Yorkshire, England
[2] Univ Leeds, Fac Biol Sci, Sch Biochem & Mol Biol, Leeds LS2 9JT, W Yorkshire, England
关键词
G-protein-coupled receptor; mutation; oocytes; three-dimensional model;
D O I
10.1042/0264-6021:3390055
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We have studied the effects of agonist and antagonist binding, agonist-induced activation and agonist-induced desensitization of the human tachykinin NK2 receptor mutated at polar residues Asn-51 [in transmembrane helix 1 (TM1)], Asp-79 (TM2) and Asn-303 (TM7), which are highly conserved in the transmembrane domain in the rhodopsin family of G-protein-coupled receptors. Wild-type and mutant receptors were expressed in both COS-1 cells and Xenopus oocytes. The results show that the N51D mutation results in a receptor which, in contrast with the wild-type receptor, is desensitized by the application of a concentration of 1 mu M of the partial agonist GR64349, indicating that the mutant is more sensitive to agonist activation than is the wild-type receptor. In addition, we show that, whereas the D79E mutant displayed activation properties similar to those of the wild-type receptor, the D79N and D79A mutants displayed a severely impaired ability to activate the calcium-dependent chloride current. This suggests that it is the negative charge at Asn-79, rather than the ability of this residue to hydrogen-bond, that is critical for the activity of the receptor. Interestingly, the placement of a negative charge at position 303 could compensate for the removal of the negative charge at position 79, since the double mutant D79N/N303D displayed activation properties similar to those of the wild-type receptor. This suggests that these two residues are functionally coupled, and may even be in close proximity in the three-dimensional structure of the human tachykinin NK2 receptor. A three-dimensional model of the receptor displaying this putative interaction is presented.
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页码:55 / 61
页数:7
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