title: Role of the extracellular loops of the thyrotropin-releasing hormone receptor: evidence for an initial interaction with thyrotropin-releasing hormone.
abstract: 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 [Ntau-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 ECLs in all G protein-coupled receptors for small ligands may be to initially contact the ligand and allow entry into a TM binding pocket.
authors.affiliation: Division of Molecular Medicine, Department of Medicine, Cornell University Medical College, New York, New York 10021, USA. jhperlma@mail.med.cornell.edu
authors.forename: J H
authors.initials: JH
authors.lastname: Perlman
authors.forename: A O
authors.initials: AO
authors.lastname: Colson
authors.forename: R
authors.initials: R
authors.lastname: Jain
authors.forename: B
authors.initials: B
authors.lastname: Czyzewski
authors.forename: L A
authors.initials: LA
authors.lastname: Cohen
authors.forename: R
authors.initials: R
authors.lastname: Osman
authors.forename: M C
authors.initials: MC
authors.lastname: Gershengorn
mesh_terms: D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D003198:Computer Simulation; D019204:GTP-Binding Proteins / Q000378:metabolism; D006860:Hydrogen Bonding; D007700:Kinetics; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D016296:Mutagenesis; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013973:Thyrotropin-Releasing Hormone / Q000378:metabolism*; D014162:Transfection / Q000235:genetics; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism
chemical_list: D008024:Ligands; D018025:Receptors, Thyrotropin-Releasing Hormone; D014443:Tyrosine; D013973:Thyrotropin-Releasing Hormone; D019204:GTP-Binding Proteins