Signal transduction for melatonin in human lymphocytes: involvement of a pertussis toxin-sensitive G protein.
We analyzed the melatonin signal transduction in human blood lymphocytes. High affinity melatonin receptors were identified by specific binding of 2-[125I]melatonin ([125I]MEL) to human lymphocyte membranes. Scatchard analysis of [125I]MEL binding revealed high affinity receptors, with a dissociation constant (Kd) of 0.45 nM and a binding capacity (Bmax) of 7.8 fmol/mg protein. Specific [125I]MEL binding was reduced markedly by GTP and its nonhydrolyzable analogues guanosine 5'-beta, gamma-imidotriphosphate (Gpp(NH)p) and guanosine 5'-O-(3-triphosphate) (GTP-gamma-S). Gpp(NH)p inhibited in a dose-dependent manner the [125I]MEL specifically bound to human lymphocyte membranes with a half-maximal inhibition (IC50) of 3.5 +/- 0.6 microM Gpp(NH)p. Treatment of human lymphocyte membranes with Gpp(NH)p increased the Kd value (Kd = 1.20 nM). Melatonin inhibited significantly and in a dose-dependent manner forskolin-stimulated cAMP production in intact human lymphocytes. Melatonin was able to stimulate diacylglycerol production in a dose-dependent manner in human lymphocyte membranes. Pertussis toxin treatment inhibited the specific [125I]MEL binding and blocked the ability of melatonin to both inhibit forskolin-stimulated cAMP production and stimulate diacylglycerol production. Pertussis toxin ADP-ribosylation and Western blot experiments demonstrated the protein expression of alpha i1/2, alpha i3/0, and beta gamma complexes of G proteins in human lymphocyte membranes. The results strongly suggest a pertussis toxin-sensitive melatonin signal transduction pathway in human lymphocytes that involves the inhibition of adenylyl cyclase and the stimulation of phospholipase C.
García-Pergañeda
A
A
Department of Medical Biochemistry and Molecular Biology, University of Seville School of Medicine, Spain.
Pozo
D
D
Guerrero
J M
JM
Calvo
J R
JR
D037361:Adenylate Cyclase Toxin; D000328:Adult; D000242:Cyclic AMP / Q000096:biosynthesis; D004075:Diglycerides / Q000096:biosynthesis; D019204:GTP-Binding Proteins / Q000187:drug effects / Q000276:immunology / Q000502:physiology*; D006150:Guanine Nucleotides / Q000494:pharmacology; D006801:Humans; D007457:Iodine Radioisotopes; D008214:Lymphocytes / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D008550:Melatonin / Q000031:analogs & derivatives / Q000276:immunology* / Q000378:metabolism / Q000494:pharmacology; D008875:Middle Aged; D037342:Pertussis Toxin; D011485:Protein Binding / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects / Q000276:immunology*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D037361:Adenylate Cyclase Toxin; D004075:Diglycerides; D006150:Guanine Nucleotides; D007457:Iodine Radioisotopes; D010566:Virulence Factors, Bordetella; C044556:2-iodomelatonin; D000242:Cyclic AMP; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D008550:Melatonin