Membrane-bound calmodulin in Xenopus laevis oocytes as a novel binding site for melatonin.
Melatonin has been suggested as a physiological antagonist of calmodulin. In this work, we have characterized melatonin binding sites in Xenopus laevis oocyte membranes. Binding of [125I]melatonin by X. laevis oocyte membranes fulfills all criteria for binding to a receptor site. Binding was dependent on time, temperature, and membrane concentration and was stable, reversible, saturable, and specific. The binding site was also pharmacologically characterized. Stoichiometric studies showed a high-affinity binding site with a Kd of 1.18 nM. These data are in close agreement with data obtained from kinetic studies (Kd=0.12 nM). In competition studies, we observed a low-affinity binding site (Kd=63.41 microM). Moreover, the binding site was characterized as calmodulin. Thus, binding was dependent on calcium and blocked by anti-CaM antibodies in a concentration-dependent manner. Calmodulin inhibitor chlorpromazine also inhibited binding of the tracer. From these results, it is suggested that membrane-bound calmodulin acts as a melatonin binding site in Xenopus laevis oocytes, where it might couple cellular activities to rhythmic circulating levels of melatonin. This hypothesis correlates with the previous findings describing melatonin as a physiological antagonist of calmodulin.
Department of Medical Biochemistry and Molecular Biology, The University of Seville School of Medicine and Virgen Macarena Hospital, 41009 Seville, Spain.carmen18@arrakis.es
M P
MP
Romero
A
A
García-Pergañeda
J M
JM
Guerrero
C
C
Osuna
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000502:physiology; D020013:Calcium Signaling / Q000502:physiology*; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002746:Chlorpromazine / Q000494:pharmacology; D004527:Egg Proteins / Q000378:metabolism*; D005260:Female; D007211:Indoles / Q000378:metabolism; D007700:Kinetics; D008550:Melatonin / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism*; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D044094:Receptors, Melatonin; D013696:Temperature; D014982:Xenopus laevis
D002147:Calmodulin; D004527:Egg Proteins; D007211:Indoles; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D044094:Receptors, Melatonin; D008550:Melatonin; D002118:Calcium; D002746:Chlorpromazine