GUANINE-NUCLEOTIDES REGULATE 2-[I-125]IODOMELATONIN BINDING-SITES IN CHICK RETINAL-PIGMENT EPITHELIUM BUT NOT IN NEURONAL RETINA

The characteristics of the binding sites labeled by the radioligand 2-[I-125]iodomelatonin were compared in chicken neuronal retina and retinal pigment epithelium (RPE). Specific binding of 2-[I-125]iodomelatonin in both sites was stable, saturable, reversible, and of high affinity. Scatchard analysis revealed an affinity constant (K(D)) of 446 +/- 55 pM and a total number of binding sites (B(max)) of 25.4 +/- 2.2 fmol/mg of protein for neuronal retina. For RPE the K(D) was 34.1 +/- 2.2 pM and the B(max) 59.5 +/- 5.2 fmol/mg of protein. Competition experiments with various melatonin analogues gave the following order of affinities: 2-iodomelatonin > 2-chloromelatonin > melatonin > 6-chloromelatonin > 6-hydroxymelatonin > N-acetylserotonin > 6-methoxyharmalan > 5-hydroxytryptamine. Linear regression of log K(i) values from neuronal retina and RPE gave a highly significant correlation (r = 0.994, n = 8; p < 0.001). GTP inhibited specific binding to RPE membranes in a concentration-dependent manner, but not in neuronal retinal membranes. The present results strongly suggest that a single type of melatonin receptor is found in neuronal retina and RPE, and that the site in RPE is coupled to a guanine nucleotide-binding regulatory protein (G protein), but that in neuronal retina is not.