MELANIN-CONCENTRATING HORMONE-BINDING SITES IN HUMAN SVK14 KERATINOCYTES

Melanin concentrating hormone (MCH) is a cyclic peptide which regulate s a broad array of functions in the mammalian brain and it may act as a paracrine factor in peripheral organs. In these studies a radiolabel ed MCH derivative, the [I-125]-[Phe(13), Tyr(19)]-MCH, was synthesized and used as a tracer to perform binding experiments. A number of huma n or rodent cell lines displayed specific binding with [I-125]-[Phe(13 ), Tyr(19)]-MCH, the highest binding capacity being observed served wi th human SVK14 keratinocytes. Saturation binding analysis with SVK14 c ells indicated about 10,000 MCH binding sites per cell and a Kd of 0.7 nM for [I-125]-[Phe(13), Tyr(19)]-MCH. Surprisingly, the iodinated [P he(13), Tyr(19)]-MCH displayed about 10-fold higher affinity (Ki simil ar to 3.0 nM) for the putative MCH receptor than the noniodinated form (Ki similar to 25-30 nM). Competition binding analyses comparing vari ous MCH-related peptides revealed a similar low binding potency for al l these peptides (Ki similar to 65-160 nM), Strikingly, rat ANP and ra t/human CNP but not rat BNP displaced [I-125][Phe(13), Tyr(15)]-MCH wi th Ki similar to 210-365 nM and may be due to topological similarities instead of partial sequence identities between MCH and some of the na triuretic peptides. However, other peptides such as CRF, alpha MSH, Ar g-vasopressin, and MGOP-peptide I did not compete with the radioligand . Finally, the molecular mass of the RICH binding sites on SVK14 cells was estimated to be 47 kDa by crosslinking and SDS-PAGE experiments. Taken together, our data revealed the widespread expression of MCH bin ding sites on mammalian cells, particularly on skin carcinoma cells. H owever, the low affinity of these sites for the native MCH and MCH-rel ated peptides as well as competitivity with ANP and CNP indicates that further biochemical and functional characterizations are needed to va lidate them as genuine physiological MCH receptors. (C) 1997 Academic Press.