Differential coupling of the extreme C-terminus of G protein alpha subunits to the G protein-coupled melatonin receptors

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

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Melatonin receptors interact with pertussis toxin-sensitive G proteins to inhibit adenylate cyclase. However, the G protein coupling profiles of melatonin receptor subtypes have not been fully characterised and alternative G protein coupling is evident. The five C-terminal residues of Galpha subunits confer coupling specificity to G protein-coupled receptors. This report outlines the use of Gas chimaeras to alter the signal output of human melatonin receptors and investigate their interaction with the C-termini of Get subunits. The Gas portion of the chimaeras confers the ability to activate adenylate cyclase leading to cyclic AMP production. Co-transfection of HEK293 cells expressing MT1, or MT2 melatonin receptors with Gas chimaeras and a cyclic AMP activated luciferase construct provided a convenient and sensitive assay system for identification of receptor recognition of Ga C-termini. Luciferase assay sensitivity was compared with measurement of cyclic AMP elevations by radioimmunoassay. Differential interactions of the melatonin receptor subtypes with Ga chimaeras were observed. Temporal and kinetic parameters of cyclic AMP responses measured by cyclic AMP radioimmunoassay varied depending on the Gas chimaeras coupled. Recognition of the C-terminal five amino acids of the Ga subunit is a requisite for coupling to a receptor, but it is not the sole determinant. (C) 2002 Elsevier Science B.V. All rights reserved.

Original languageEnglish
Pages (from-to)185-192
Number of pages8
JournalBiochimica et Biophysica Acta. Molecular Cell Research
Volume1592
Issue number2
Early online date16 Sep 2002
DOIs
Publication statusPublished - 21 Oct 2002

Keywords

  • melatonin receptor
  • G protein-coupled receptor
  • G alpha chimaera
  • xenopus dermal melanophores
  • ovine pars tuberalis
  • 2-<I-125>iodomelatonin binding
  • signal-transduction
  • ligand-binding
  • identification
  • activation
  • expression
  • residues
  • MEL1A

Cite this

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title = "Differential coupling of the extreme C-terminus of G protein alpha subunits to the G protein-coupled melatonin receptors",
abstract = "Melatonin receptors interact with pertussis toxin-sensitive G proteins to inhibit adenylate cyclase. However, the G protein coupling profiles of melatonin receptor subtypes have not been fully characterised and alternative G protein coupling is evident. The five C-terminal residues of Galpha subunits confer coupling specificity to G protein-coupled receptors. This report outlines the use of Gas chimaeras to alter the signal output of human melatonin receptors and investigate their interaction with the C-termini of Get subunits. The Gas portion of the chimaeras confers the ability to activate adenylate cyclase leading to cyclic AMP production. Co-transfection of HEK293 cells expressing MT1, or MT2 melatonin receptors with Gas chimaeras and a cyclic AMP activated luciferase construct provided a convenient and sensitive assay system for identification of receptor recognition of Ga C-termini. Luciferase assay sensitivity was compared with measurement of cyclic AMP elevations by radioimmunoassay. Differential interactions of the melatonin receptor subtypes with Ga chimaeras were observed. Temporal and kinetic parameters of cyclic AMP responses measured by cyclic AMP radioimmunoassay varied depending on the Gas chimaeras coupled. Recognition of the C-terminal five amino acids of the Ga subunit is a requisite for coupling to a receptor, but it is not the sole determinant. (C) 2002 Elsevier Science B.V. All rights reserved.",
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author = "Janice Drew and Perry Barrett and S Conway and P Delagrange and Morgan, {Peter John}",
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T1 - Differential coupling of the extreme C-terminus of G protein alpha subunits to the G protein-coupled melatonin receptors

AU - Drew, Janice

AU - Barrett, Perry

AU - Conway, S

AU - Delagrange, P

AU - Morgan, Peter John

PY - 2002/10/21

Y1 - 2002/10/21

N2 - Melatonin receptors interact with pertussis toxin-sensitive G proteins to inhibit adenylate cyclase. However, the G protein coupling profiles of melatonin receptor subtypes have not been fully characterised and alternative G protein coupling is evident. The five C-terminal residues of Galpha subunits confer coupling specificity to G protein-coupled receptors. This report outlines the use of Gas chimaeras to alter the signal output of human melatonin receptors and investigate their interaction with the C-termini of Get subunits. The Gas portion of the chimaeras confers the ability to activate adenylate cyclase leading to cyclic AMP production. Co-transfection of HEK293 cells expressing MT1, or MT2 melatonin receptors with Gas chimaeras and a cyclic AMP activated luciferase construct provided a convenient and sensitive assay system for identification of receptor recognition of Ga C-termini. Luciferase assay sensitivity was compared with measurement of cyclic AMP elevations by radioimmunoassay. Differential interactions of the melatonin receptor subtypes with Ga chimaeras were observed. Temporal and kinetic parameters of cyclic AMP responses measured by cyclic AMP radioimmunoassay varied depending on the Gas chimaeras coupled. Recognition of the C-terminal five amino acids of the Ga subunit is a requisite for coupling to a receptor, but it is not the sole determinant. (C) 2002 Elsevier Science B.V. All rights reserved.

AB - Melatonin receptors interact with pertussis toxin-sensitive G proteins to inhibit adenylate cyclase. However, the G protein coupling profiles of melatonin receptor subtypes have not been fully characterised and alternative G protein coupling is evident. The five C-terminal residues of Galpha subunits confer coupling specificity to G protein-coupled receptors. This report outlines the use of Gas chimaeras to alter the signal output of human melatonin receptors and investigate their interaction with the C-termini of Get subunits. The Gas portion of the chimaeras confers the ability to activate adenylate cyclase leading to cyclic AMP production. Co-transfection of HEK293 cells expressing MT1, or MT2 melatonin receptors with Gas chimaeras and a cyclic AMP activated luciferase construct provided a convenient and sensitive assay system for identification of receptor recognition of Ga C-termini. Luciferase assay sensitivity was compared with measurement of cyclic AMP elevations by radioimmunoassay. Differential interactions of the melatonin receptor subtypes with Ga chimaeras were observed. Temporal and kinetic parameters of cyclic AMP responses measured by cyclic AMP radioimmunoassay varied depending on the Gas chimaeras coupled. Recognition of the C-terminal five amino acids of the Ga subunit is a requisite for coupling to a receptor, but it is not the sole determinant. (C) 2002 Elsevier Science B.V. All rights reserved.

KW - melatonin receptor

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KW - xenopus dermal melanophores

KW - ovine pars tuberalis

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KW - signal-transduction

KW - ligand-binding

KW - identification

KW - activation

KW - expression

KW - residues

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