Role of the extracellular loops of the thyrotropin-releasing hormone receptor: Evidence for an initial interaction with thyrotropin-releasing hormone

被引:45
作者
Perlman, JH
Colson, AO
Jain, R
Czyzewski, B
Cohen, LA
Osman, R
Gershengorn, MC
机构
[1] Cornell Univ, Coll Med, Dept Med, Div Mol Med, New York, NY 10021 USA
[2] CUNY Mt Sinai Sch Med, Dept Physiol & Biophys, New York, NY 10029 USA
[3] NIDDKD, Bioorgan Chem Lab, NIH, Bethesda, MD 20892 USA
关键词
D O I
10.1021/bi9713310
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
Thyrotropin-releasing hormone (TRH), like most small ligands, appears to bind within the seven transmembrane-spanning helices (TMs) of its G protein-coupled receptor (TRH-R), A role for the extracellular loops (ECLs) of TRH-R has not been established, We substituted residues in the ECLs of TRH-R and show that Tyr-181 is important for high-affinity binding because its substitution leads to a 3700-fold lowering of the estimated affinity compared to wild-type TRH-R. Using TRH analogues, we provide evidence that there is a specific interaction between Tyr-181 in ECL-2 and the pyroGlu moiety of TRH, It was previously suggested that the pyroGlu of TRH may interact with Asn-110 in TM-3 and with Asn-289 in ECL-3; N110A and N289A TRH-Rs exhibit similar apparent affinities that are only 20-30-fold lower than wild-type TRH-R, To better understand these findings, we analyzed a computer-generated model which predicts that the ECLs form an entry channel into the TRH-R TM bundle, that Tyr-181 projects into this channel and that the pyroGlu of TRH cannot simultaneously interact with residues in the TMs and ECLs. Kinetic analysis showed that the association rate of [N-tau-methyl-His]TRH with N289A TRH-R is slower than with wild-type TRH-R and largely accounts for the lower apparent affinity; the association rate with N110A TRH-R is similar to that of wild-type TRH-R. These data are consistent with the idea that there are initial interactions between TRH and the residues of a putative entry channel of TRH-R. We suggest that a role of the EC Ls in all G protein-coupled receptors for small ligands maybe to initially contact the ligand and allow entry into a TM binding pocket.
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页码:15670 / 15676
页数:7
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