Melanin-concentrating hormone binding sites in human SVK14 keratinocytes.
Melanin concentrating hormone (MCH) is a cyclic peptide which regulates a broad array of functions in the mammalian brain and it may act as a paracrine factor in peripheral organs. In these studies a radiolabeled MCH derivative, the [125I]-[Phe13, Tyr19]-MCH, was synthesized and used as a tracer to perform binding experiments. A number of human or rodent cell lines displayed specific binding with [125I]-[Phe13, Tyr19]-MCH, the highest binding capacity being observed with human SVK14 keratinocytes. Saturation binding analysis with SVK14 cells indicated about 10,000 MCH binding sites per cell and a Kd of 0.7 nM for [125I]-[Phe13, Tyr19]-MCH. Surprisingly, the iodinated [Phe13, Tyr19]-MCH displayed about 10-fold higher affinity (Ki approximately 3.0 nM) for the putative MCH receptor than the noniodinated form (Ki approximately 25-30 nM). Competition binding analyses comparing various MCH-related peptides revealed a similar low binding potency for all these peptides (Ki approximately 65-160 nM). Strikingly, rat ANP and rat/human CNP but not rat BNP displaced [125I]-[Phe13, Tyr15]-MCH with Ki approximately 210-365 nM and may be due to topological similarities instead of partial sequence identities between MCH and some of the natriuretic peptides. However, other peptides such as CRF, alpha MSH, Arg-vasopressin, and MGOP-peptide I did not compete with the radioligand. Finally, the molecular mass of the MCH 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 binding sites on mammalian cells, particularly on skin carcinoma cells. However, the low affinity of these sites for the native MCH and MCH-related peptides as well as competitivity with ANP and CNP indicates that further biochemical and functional characterizations are needed to validate them as genuine physiological MCH receptors.
Burgaud
J L
JL
Institut de Pharmacologie Moléculaire et Cellulaire, CNRS UPR 411, Valbonne, France.
Poosti
R
R
Fehrentz
J A
JA
Martinez
J
J
Nahon
J L
JL
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002294:Carcinoma, Squamous Cell; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D007028:Hypothalamic Hormones / Q000378:metabolism*; D007457:Iodine Radioisotopes; D015603:Keratinocytes / Q000378:metabolism*; D008543:Melanins / Q000378:metabolism*; D008545:Melanoma; D008969:Molecular Sequence Data; D009457:Neuroglia; D016716:PC12 Cells; D010649:Phenylalanine / Q000378:metabolism; D010907:Pituitary Hormones / Q000378:metabolism*; D011869:Radioligand Assay; D051381:Rats; D011979:Receptors, Pituitary Hormone / Q000737:chemistry; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism
D007028:Hypothalamic Hormones; D007457:Iodine Radioisotopes; D008543:Melanins; D010907:Pituitary Hormones; D011979:Receptors, Pituitary Hormone; C092274:melanin-concentrating hormone receptor; D014443:Tyrosine; D010649:Phenylalanine; C019463:melanin-concentrating hormone