(D-(p-benzoylphenylalanine)13, tyrosine19)-melanin-concentrating hormone, a potent analogue for MCH receptor crosslinking.
A photoreactive analogue of human melanin-concentrating hormone was designed, [D-Bpa13,Tyr19-MCH, containing the D-enantiomer of photolabile p-benzoylphenylalanine (Bpa) in position 13 and tyrosine for radioiodination in position 19. The linear peptide was synthesized by the continuous-flow solid-phase methodology using Fmoc-strategy and PEG-PS resins, purified to homogeneity and cyclized by iodine oxidation. Radioiodination of [D-Bpa13,Tyr19]-MCH at its Tyr19 residue was carried out enzymatically using solid-phase bound glucose oxidase/lactoperoxidase, followed by purification on a reversed-phase mini-column and HPLC. Saturation binding analysis of [125I]-[D-Bpa13,Tyr19]-MCH with G4F-7 mouse melanoma cells gave a K(D) of 2.2+/-0.2 x 10(-10) mol/l and a B(max) of 1047+/-50 receptors/cell. Competition binding analysis showed that MCH and rANF(1-28) displace [125I]-[D-Bpa13,Tyr19]-MCH from the MCH binding sites on G4F-7 cells whereas alpha-MSH has no effect. Receptor crosslinking by UV-irradiation of G4F-7 cells in the presence of [125I]-[D-Bpa13,Tyr19]-MCH followed by SDS-polyacrylamide gel electrophoresis and autoradiography yielded a band of 45-50 kDa. Identical crosslinked bands were also detected in B16-F1 and G4F mouse melanoma cells, in RE and D10 human melanoma cells as well as in COS-7 cells. Weak staining was found in rat PC12 phaeochromocytoma and Chinese hamster ovary cells. No crosslinking was detected in human MP fibroblasts. These data demonstrate that [125I]-[D-Bpa13,Tyr19]-MCH is a versatile photocrosslinking analogue of MCH suitable to identify MCH receptors in different cells and tissues; the MCH receptor in these cells appears to have the size of a G protein-coupled receptor, most likely with a varying degree of glycosylation.
Drozdz
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Department of Research (ZLF), University Hospital and University Children's Hospital, Basel, Switzerland.
Hintermann
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Tanner
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Zumsteg
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Eberle
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D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006801:Humans; D007457:Iodine Radioisotopes / Q000737:chemistry; D008543:Melanins / Q000138:chemical synthesis / Q000737:chemistry*; D008969:Molecular Sequence Data; D019920:Photoaffinity Labels; D011869:Radioligand Assay; D011979:Receptors, Pituitary Hormone / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured
C120437:(4-benzoylphenylalanyl(13),tyrosyl(19))-melanin; D007457:Iodine Radioisotopes; D008543:Melanins; D019920:Photoaffinity Labels; D011979:Receptors, Pituitary Hormone; C092274:melanin-concentrating hormone receptor