Dual signaling of human Mel1a melatonin receptors via G(i2), G(i3), and G(q/11) proteins

L Brydon, F Roka, L Petit, P de Coppet, M Tissot, Perry Barrett, Peter John Morgan, C Nanoff, A D Strosberg, R Jockers

Research output: Contribution to journalArticle

216 Citations (Scopus)

Abstract

Mel 1a melatonin receptors belong to the superfamily of guanine nucleotide-binding regulatory protein (G protein)-coupled receptors. So far, interest in Mel la receptor signaling has focused mainly on the modulation of the adenylyl cyclase pathway via pertussis toxin (PTX)-sensitive G proteins. To further investigate signaling of the human Mel 1a receptor, we have developed an antibody directed against the C terminus of this receptor. This antibody detected the Mel 1a receptor as a protein with an apparent molecular mass of approximately 60 kDa in immunoblots after separation by SDS-PAGE. It also specifically precipitated the 2-[I-125]iodomelatonin (I-125-Mel)-labeled receptor from Mel 1a-transfected HEK 293 cells. Coprecipitation experiments showed that G(i2), G(i3), and G(q/11) proteins couple to the Mel 1a receptor in an agonist-dependent and guanine nucleotide-sensitive manner. Coupling was selective since other G proteins present in HEK 293 cells, (G(i1), G(o), G(s), G(z), and G(12)) were not detected in receptor complexes. Coupling of the Mel 1a receptor to G(i) and G(q) was confirmed by inhibition of high-affinity I-125-Mel binding to receptors with subtype-selective G protein alpha-subunit antibodies. G(i2) and/or G(i3) mediated adenylyl cyclase inhibition while G(q/11) induced a transient elevation in cytosolic calcium concentrations in HEK 293 cells stably expressing Mel 1a receptors. Melatonin-induced cytosolic calcium mobilization via PTX-insensitive G proteins was confirmed in primary cultures of ovine pars tuberalis cells endogenously expressing Mel 1a receptors. In conclusion, we report the development of the first antibody recognizing the cloned human Mel 1a melatonin receptor protein. We show that Mel 1a receptors functionally couple to both PTX-sensitive and PTX-insensitive G proteins. The previously unknown signaling of Mel 1a receptors through G(q/11) widens the spectrum of potential targets for melatonin.

Original languageEnglish
Pages (from-to)2025-2038
Number of pages14
JournalMolecular Endocrinology
Volume13
Issue number12
DOIs
Publication statusPublished - Dec 1999

Keywords

  • ovine pars-tuberalis
  • suprachiasmatic circadian clock
  • alpha-subunits
  • kinase-C
  • phospholipase-C
  • dopamine-receptors
  • phorbol ester
  • cells
  • expression
  • activation

Cite this

Dual signaling of human Mel1a melatonin receptors via G(i2), G(i3), and G(q/11) proteins. / Brydon, L ; Roka, F ; Petit, L ; de Coppet, P ; Tissot, M ; Barrett, Perry; Morgan, Peter John; Nanoff, C ; Strosberg, A D ; Jockers, R .

In: Molecular Endocrinology, Vol. 13, No. 12, 12.1999, p. 2025-2038.

Research output: Contribution to journalArticle

Brydon, L, Roka, F, Petit, L, de Coppet, P, Tissot, M, Barrett, P, Morgan, PJ, Nanoff, C, Strosberg, AD & Jockers, R 1999, 'Dual signaling of human Mel1a melatonin receptors via G(i2), G(i3), and G(q/11) proteins', Molecular Endocrinology, vol. 13, no. 12, pp. 2025-2038. https://doi.org/10.1210/me.13.12.2025
Brydon, L ; Roka, F ; Petit, L ; de Coppet, P ; Tissot, M ; Barrett, Perry ; Morgan, Peter John ; Nanoff, C ; Strosberg, A D ; Jockers, R . / Dual signaling of human Mel1a melatonin receptors via G(i2), G(i3), and G(q/11) proteins. In: Molecular Endocrinology. 1999 ; Vol. 13, No. 12. pp. 2025-2038.
@article{8a80670684de45719c98053adff67a38,
title = "Dual signaling of human Mel1a melatonin receptors via G(i2), G(i3), and G(q/11) proteins",
abstract = "Mel 1a melatonin receptors belong to the superfamily of guanine nucleotide-binding regulatory protein (G protein)-coupled receptors. So far, interest in Mel la receptor signaling has focused mainly on the modulation of the adenylyl cyclase pathway via pertussis toxin (PTX)-sensitive G proteins. To further investigate signaling of the human Mel 1a receptor, we have developed an antibody directed against the C terminus of this receptor. This antibody detected the Mel 1a receptor as a protein with an apparent molecular mass of approximately 60 kDa in immunoblots after separation by SDS-PAGE. It also specifically precipitated the 2-[I-125]iodomelatonin (I-125-Mel)-labeled receptor from Mel 1a-transfected HEK 293 cells. Coprecipitation experiments showed that G(i2), G(i3), and G(q/11) proteins couple to the Mel 1a receptor in an agonist-dependent and guanine nucleotide-sensitive manner. Coupling was selective since other G proteins present in HEK 293 cells, (G(i1), G(o), G(s), G(z), and G(12)) were not detected in receptor complexes. Coupling of the Mel 1a receptor to G(i) and G(q) was confirmed by inhibition of high-affinity I-125-Mel binding to receptors with subtype-selective G protein alpha-subunit antibodies. G(i2) and/or G(i3) mediated adenylyl cyclase inhibition while G(q/11) induced a transient elevation in cytosolic calcium concentrations in HEK 293 cells stably expressing Mel 1a receptors. Melatonin-induced cytosolic calcium mobilization via PTX-insensitive G proteins was confirmed in primary cultures of ovine pars tuberalis cells endogenously expressing Mel 1a receptors. In conclusion, we report the development of the first antibody recognizing the cloned human Mel 1a melatonin receptor protein. We show that Mel 1a receptors functionally couple to both PTX-sensitive and PTX-insensitive G proteins. The previously unknown signaling of Mel 1a receptors through G(q/11) widens the spectrum of potential targets for melatonin.",
keywords = "ovine pars-tuberalis, suprachiasmatic circadian clock, alpha-subunits, kinase-C, phospholipase-C, dopamine-receptors, phorbol ester, cells, expression, activation",
author = "L Brydon and F Roka and L Petit and {de Coppet}, P and M Tissot and Perry Barrett and Morgan, {Peter John} and C Nanoff and Strosberg, {A D} and R Jockers",
year = "1999",
month = "12",
doi = "10.1210/me.13.12.2025",
language = "English",
volume = "13",
pages = "2025--2038",
journal = "Molecular Endocrinology",
issn = "0888-8809",
publisher = "The Endocrine Society",
number = "12",

}

TY - JOUR

T1 - Dual signaling of human Mel1a melatonin receptors via G(i2), G(i3), and G(q/11) proteins

AU - Brydon, L

AU - Roka, F

AU - Petit, L

AU - de Coppet, P

AU - Tissot, M

AU - Barrett, Perry

AU - Morgan, Peter John

AU - Nanoff, C

AU - Strosberg, A D

AU - Jockers, R

PY - 1999/12

Y1 - 1999/12

N2 - Mel 1a melatonin receptors belong to the superfamily of guanine nucleotide-binding regulatory protein (G protein)-coupled receptors. So far, interest in Mel la receptor signaling has focused mainly on the modulation of the adenylyl cyclase pathway via pertussis toxin (PTX)-sensitive G proteins. To further investigate signaling of the human Mel 1a receptor, we have developed an antibody directed against the C terminus of this receptor. This antibody detected the Mel 1a receptor as a protein with an apparent molecular mass of approximately 60 kDa in immunoblots after separation by SDS-PAGE. It also specifically precipitated the 2-[I-125]iodomelatonin (I-125-Mel)-labeled receptor from Mel 1a-transfected HEK 293 cells. Coprecipitation experiments showed that G(i2), G(i3), and G(q/11) proteins couple to the Mel 1a receptor in an agonist-dependent and guanine nucleotide-sensitive manner. Coupling was selective since other G proteins present in HEK 293 cells, (G(i1), G(o), G(s), G(z), and G(12)) were not detected in receptor complexes. Coupling of the Mel 1a receptor to G(i) and G(q) was confirmed by inhibition of high-affinity I-125-Mel binding to receptors with subtype-selective G protein alpha-subunit antibodies. G(i2) and/or G(i3) mediated adenylyl cyclase inhibition while G(q/11) induced a transient elevation in cytosolic calcium concentrations in HEK 293 cells stably expressing Mel 1a receptors. Melatonin-induced cytosolic calcium mobilization via PTX-insensitive G proteins was confirmed in primary cultures of ovine pars tuberalis cells endogenously expressing Mel 1a receptors. In conclusion, we report the development of the first antibody recognizing the cloned human Mel 1a melatonin receptor protein. We show that Mel 1a receptors functionally couple to both PTX-sensitive and PTX-insensitive G proteins. The previously unknown signaling of Mel 1a receptors through G(q/11) widens the spectrum of potential targets for melatonin.

AB - Mel 1a melatonin receptors belong to the superfamily of guanine nucleotide-binding regulatory protein (G protein)-coupled receptors. So far, interest in Mel la receptor signaling has focused mainly on the modulation of the adenylyl cyclase pathway via pertussis toxin (PTX)-sensitive G proteins. To further investigate signaling of the human Mel 1a receptor, we have developed an antibody directed against the C terminus of this receptor. This antibody detected the Mel 1a receptor as a protein with an apparent molecular mass of approximately 60 kDa in immunoblots after separation by SDS-PAGE. It also specifically precipitated the 2-[I-125]iodomelatonin (I-125-Mel)-labeled receptor from Mel 1a-transfected HEK 293 cells. Coprecipitation experiments showed that G(i2), G(i3), and G(q/11) proteins couple to the Mel 1a receptor in an agonist-dependent and guanine nucleotide-sensitive manner. Coupling was selective since other G proteins present in HEK 293 cells, (G(i1), G(o), G(s), G(z), and G(12)) were not detected in receptor complexes. Coupling of the Mel 1a receptor to G(i) and G(q) was confirmed by inhibition of high-affinity I-125-Mel binding to receptors with subtype-selective G protein alpha-subunit antibodies. G(i2) and/or G(i3) mediated adenylyl cyclase inhibition while G(q/11) induced a transient elevation in cytosolic calcium concentrations in HEK 293 cells stably expressing Mel 1a receptors. Melatonin-induced cytosolic calcium mobilization via PTX-insensitive G proteins was confirmed in primary cultures of ovine pars tuberalis cells endogenously expressing Mel 1a receptors. In conclusion, we report the development of the first antibody recognizing the cloned human Mel 1a melatonin receptor protein. We show that Mel 1a receptors functionally couple to both PTX-sensitive and PTX-insensitive G proteins. The previously unknown signaling of Mel 1a receptors through G(q/11) widens the spectrum of potential targets for melatonin.

KW - ovine pars-tuberalis

KW - suprachiasmatic circadian clock

KW - alpha-subunits

KW - kinase-C

KW - phospholipase-C

KW - dopamine-receptors

KW - phorbol ester

KW - cells

KW - expression

KW - activation

U2 - 10.1210/me.13.12.2025

DO - 10.1210/me.13.12.2025

M3 - Article

VL - 13

SP - 2025

EP - 2038

JO - Molecular Endocrinology

JF - Molecular Endocrinology

SN - 0888-8809

IS - 12

ER -