Guanine nucleotide-binding protein regulation of melatonin receptors in lizard brain.

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Abstract
Melatonin receptors were identified and characterized in crude membrane preparations from lizard brain by using 125I-labeled melatonin (125I-Mel), a potent melatonin agonist. 125I-Mel binding sites were saturable; Scatchard analysis revealed high-affinity and lower affinity binding sites, with apparent Kd of 2.3 .+-. 1.0 .times. 10-11 M and 2.06 .+-. 0.43 .times. 10-10 M, respectively. Binding was reversible and inhibited by melatonin related analogs but not by serotonin or norepinephrine. Treatment of crude membranes with the nonhydrolyable GTP analog guanosine 5''-[.gamma.-thio]triphosphate (GTP[.gamma.S]), significantly reduced the number of high-affinity receptors and increased the dissociation rate of 125I-Mel from its receptor. Furthermore, GTP[.gamma.S] treatment of ligand-receptor complexes solubilized by Triton X-100 also led to a rapid dissociation of 125I-Mel from solubilized ligand-receptor complexes. Gel filtration chromatography of solubilized ligand-receptor complexes revealed two major peaks of radioactivity corresponding to Mr > 400,000 and Mr ca. 110,000. This elution profile was markedly altered by pretreatment with GTP[.gamma.S] before solubilization; only the Mr 110,000 peak was present in GTP[.gamma.S]-pretreated membranes. The results strongly suggest that 125I-Mel binding sites in lizard brain are melatonin receptors, with agonist-promoted guanine nucleotide-binding protein (G protein) couplong and that the apparent molecular size of receptors uncoupled from G proteins is about 110,000.