(D-(p-benzoylphenylalanine)(13), tyrosine(19))-melanin-concentrating hormone, a potent analogue for MCH receptor crosslinking

A photoreactive analogue of human melanin-concentrating hormone was designe d, [D-Bpa(13), Tyr(19)]-MCH. containing the D-enantiomer of photolabile p-b enzoylphenylalanine (Bpa) in position 13 and tyrosine for radioiodination i n position 19. The linear peptide was synthesized by the continuous-flow so lid-phase methodology using Fmoc-strategy and PEG-PS resins, purified to ho mogeneity and cyclized by iodine oxidation. Radioiodination of [D-Bpa(13),T yr(19)]-MCH at its Tyr(19) residue was carried out enzymatically using soli d-phase bound glucose oxidase/lactoperoxidase, followed by purification on a reversed-phase mini-column and HPLC. Saturation binding analysis of [I-12 5]-[D-Bpa(13),Tyr(19)]-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. Compet ition binding analysis showed that MCH and rANF(1-28) displace [I-125]-[D-B pa(13),Tyr(19)]-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 [I-125]-[D-Bpa(13),Tyr(19)]-MCH followed by SDS-polyacry lamide gel electrophoresis and autoradiography yielded a band of 45-50 kDa. identical crosslinked bands were also detected in B16-F1 and G4F mouse mel anoma 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 w ary cells. No crosslinking was detected in human MP fibroblasts. These data demonstrate that [I-125]-[D-Bpa(13),Tyr(19)]-MCH is a versatile photocross linking analogue of MCH suitable to identify MCH receptors in different cel ls 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 glycosy lation. Copyright (C) 1999 European Peptide Society and John Wiley & Sons, Ltd.