Tight Association of the Human Mel1a-Melatonin Receptor and Gi: Precoupling and Constitutive Activity

  1. Florian Roka1,
  2. Lena Brydon2,
  3. Maria Waldhoer1,
  4. A. Donny Strosberg2,
  5. Michael Freissmuth1,
  6. Ralf Jockers2 and
  7. Christian Nanoff1
  1. 1Institute of Pharmacology, University of Vienna, Vienna, Austria (F.R., M.W., M.F., C.N.), and 2Institut Cochin de Genétique Moléculaire, Paris, France (L.B., A.D.S., R.J.)

    Abstract

    If stably expressed in human embryonic kidney (HEK)293 cells, the human Mel1a-melatonin receptor activates Gi-dependent, pertussis toxin-sensitive signaling pathways, i.e., inhibition of adenylyl cyclase and stimulation of phospholipase Cβ; the latter on condition that Gq is coactivated. The antagonist luzindole blocks the effects of melatonin and acts as an inverse agonist at the Mel1a receptor in both intact cells and isolated membranes. This suggests that the Mel1areceptor is endowed with constitutive activity, a finding confirmed on reconstitution of the Mel1a receptor with Gi. Because the receptor density is in the physiological range, constitutive activity is not an artifact arising from overexpression of the receptor. In addition, the following findings indicate that the Mel1a receptor forms a very tight complex with Gi which can be observed both in the presence and absence of an agonist. 1) In intact cells and in membranes, high-affinity agonist binding is resistant to the destabilizing effect of guanine nucleotides. 2) The ability to bind an agonist with high affinity is preserved even after exposure of the cells to pertussis toxin, because a fraction of Gi is inaccessible to the toxin in cells expressing Mel1a receptors (but not the A1-adenosine receptor, another Gi-coupled receptor). 3) An antiserum directed against the Mel1areceptor coprecipitates Gi even in the absence of an agonist. We therefore conclude that the Mel1a receptor is tightly precoupled and that its constitutive activity may play a role in pacing the biological clock, an action known to involve the melatonin receptors in the suprachiasmatic nucleus.

    Footnotes

    • Send reprint requests to: Dr. Christian Nanoff, Institute of Pharmacology, Vienna University, Wahringer Str. 13A, A-1090 Vienna, Austria. E-mail: christian.nanoff{at}univie.ac.at

    • This work was supported by grants from the Austrian Science Foundation (FWF) to C.N. (P-12125) and to M.F. (P-12079), by a grant from the Institut de Recherches Internationales de Servier, and by the European Union-sponsored concerted action European Network for Biological Signal Transduction.

    • Abbreviations:
      PTX
      pertussis toxin
      OG
      octylglucoside
      iodomelatonin
      2-[125I]iodomelatonin
      HEK
      human embryonic kidney
      TCA
      trichloroacetic acid
      PCA
      perchloroacetic acid
      IP
      inositol phosphate
      NAD
      nicotinamide adenine dinucleotide
      R/G
      receptor/G protein
      • Received April 28, 1999.
      • Accepted August 11, 1999.
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    1. Molecular Pharmacology November 1, 1999 vol. 56 no. 5 1014-1024
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