Chimeric melatonin mt(1) and melatonin-related receptors Identification of domains and residues participating in ligand binding and receptor activation of the melatonin mt(1) receptor

S Conway, Janice Drew, E S Mowat, Perry Barrett, P Delagrange, Peter John Morgan

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42 Citations (Scopus)

Abstract

Melatonin receptors bind and become activated by melatonin. The melatonin-related receptor, despite sharing considerable amino acid sequence identity with melatonin receptors, does not bind melatonin and is currently an orphan G protein-coupled receptor. To investigate the structure and function of both receptors, we engineered a series of 14 chimeric receptor constructs, allowing us to determine the relative contribution of each transmembrane domain to Ligand binding and receptor function. Results identified that when sequences encoding transmembrane domains 1, 2, 3, 5, or 7 of the melatonin mt, receptor were replaced by the corresponding domains of the melatonin-related receptor, the resultant chimeric receptors all displayed specific 2-[I-125]iodomelatonin binding, Replacement of sequences incorporating transmembrane domains 4 or 6, however, resulted in chimeric receptors that displayed no detectable 2-[I-125]iodomelatonin binding. The subsequent testing of a "reverse" chimeric receptor in which sequences encoding transmembrane domains 4 and 6 of the melatonin-related receptor were replaced by the corresponding melatonin mt, receptor sequences identified specific 2-[I-125]iodomelatonin binding and melatonin-mediated modulation of cyclic AMP levels. To further investigate these findings, site-directed mutagenesis was performed on residues within transmembrane domain 6 of the melatonin mt, receptor. This identified Gly(258) (Gly(6.55)) as a critical residue required for high affinity ligand binding and receptor function.

Original languageEnglish
Pages (from-to)20602-20609
Number of pages8
JournalThe Journal of Biological Chemistry
Volume275
Issue number27
DOIs
Publication statusPublished - 7 Jul 2000

Keywords

  • beta-adrenergic-receptor
  • protein-coupled receptors
  • gene synthesis
  • beta-2-adrenergic receptors
  • molecular-cloning
  • adenylate-cyclase
  • expression
  • rhodopsin
  • inhibition
  • construction

Cite this

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title = "Chimeric melatonin mt(1) and melatonin-related receptors Identification of domains and residues participating in ligand binding and receptor activation of the melatonin mt(1) receptor",
abstract = "Melatonin receptors bind and become activated by melatonin. The melatonin-related receptor, despite sharing considerable amino acid sequence identity with melatonin receptors, does not bind melatonin and is currently an orphan G protein-coupled receptor. To investigate the structure and function of both receptors, we engineered a series of 14 chimeric receptor constructs, allowing us to determine the relative contribution of each transmembrane domain to Ligand binding and receptor function. Results identified that when sequences encoding transmembrane domains 1, 2, 3, 5, or 7 of the melatonin mt, receptor were replaced by the corresponding domains of the melatonin-related receptor, the resultant chimeric receptors all displayed specific 2-[I-125]iodomelatonin binding, Replacement of sequences incorporating transmembrane domains 4 or 6, however, resulted in chimeric receptors that displayed no detectable 2-[I-125]iodomelatonin binding. The subsequent testing of a {"}reverse{"} chimeric receptor in which sequences encoding transmembrane domains 4 and 6 of the melatonin-related receptor were replaced by the corresponding melatonin mt, receptor sequences identified specific 2-[I-125]iodomelatonin binding and melatonin-mediated modulation of cyclic AMP levels. To further investigate these findings, site-directed mutagenesis was performed on residues within transmembrane domain 6 of the melatonin mt, receptor. This identified Gly(258) (Gly(6.55)) as a critical residue required for high affinity ligand binding and receptor function.",
keywords = "beta-adrenergic-receptor, protein-coupled receptors, gene synthesis, beta-2-adrenergic receptors, molecular-cloning, adenylate-cyclase, expression, rhodopsin, inhibition, construction",
author = "S Conway and Janice Drew and Mowat, {E S} and Perry Barrett and P Delagrange and Morgan, {Peter John}",
year = "2000",
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T1 - Chimeric melatonin mt(1) and melatonin-related receptors Identification of domains and residues participating in ligand binding and receptor activation of the melatonin mt(1) receptor

AU - Conway, S

AU - Drew, Janice

AU - Mowat, E S

AU - Barrett, Perry

AU - Delagrange, P

AU - Morgan, Peter John

PY - 2000/7/7

Y1 - 2000/7/7

N2 - Melatonin receptors bind and become activated by melatonin. The melatonin-related receptor, despite sharing considerable amino acid sequence identity with melatonin receptors, does not bind melatonin and is currently an orphan G protein-coupled receptor. To investigate the structure and function of both receptors, we engineered a series of 14 chimeric receptor constructs, allowing us to determine the relative contribution of each transmembrane domain to Ligand binding and receptor function. Results identified that when sequences encoding transmembrane domains 1, 2, 3, 5, or 7 of the melatonin mt, receptor were replaced by the corresponding domains of the melatonin-related receptor, the resultant chimeric receptors all displayed specific 2-[I-125]iodomelatonin binding, Replacement of sequences incorporating transmembrane domains 4 or 6, however, resulted in chimeric receptors that displayed no detectable 2-[I-125]iodomelatonin binding. The subsequent testing of a "reverse" chimeric receptor in which sequences encoding transmembrane domains 4 and 6 of the melatonin-related receptor were replaced by the corresponding melatonin mt, receptor sequences identified specific 2-[I-125]iodomelatonin binding and melatonin-mediated modulation of cyclic AMP levels. To further investigate these findings, site-directed mutagenesis was performed on residues within transmembrane domain 6 of the melatonin mt, receptor. This identified Gly(258) (Gly(6.55)) as a critical residue required for high affinity ligand binding and receptor function.

AB - Melatonin receptors bind and become activated by melatonin. The melatonin-related receptor, despite sharing considerable amino acid sequence identity with melatonin receptors, does not bind melatonin and is currently an orphan G protein-coupled receptor. To investigate the structure and function of both receptors, we engineered a series of 14 chimeric receptor constructs, allowing us to determine the relative contribution of each transmembrane domain to Ligand binding and receptor function. Results identified that when sequences encoding transmembrane domains 1, 2, 3, 5, or 7 of the melatonin mt, receptor were replaced by the corresponding domains of the melatonin-related receptor, the resultant chimeric receptors all displayed specific 2-[I-125]iodomelatonin binding, Replacement of sequences incorporating transmembrane domains 4 or 6, however, resulted in chimeric receptors that displayed no detectable 2-[I-125]iodomelatonin binding. The subsequent testing of a "reverse" chimeric receptor in which sequences encoding transmembrane domains 4 and 6 of the melatonin-related receptor were replaced by the corresponding melatonin mt, receptor sequences identified specific 2-[I-125]iodomelatonin binding and melatonin-mediated modulation of cyclic AMP levels. To further investigate these findings, site-directed mutagenesis was performed on residues within transmembrane domain 6 of the melatonin mt, receptor. This identified Gly(258) (Gly(6.55)) as a critical residue required for high affinity ligand binding and receptor function.

KW - beta-adrenergic-receptor

KW - protein-coupled receptors

KW - gene synthesis

KW - beta-2-adrenergic receptors

KW - molecular-cloning

KW - adenylate-cyclase

KW - expression

KW - rhodopsin

KW - inhibition

KW - construction

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