Synthesis and lodination of human (phenylalanine¹³, tyrosine¹⁹) melanin‐concentrating hormone for radioreceptor assay

Abstract

An analogue of human melanin-concentrating hormone (MCH) suitable for radioiodination was designed in which Tyr13 and Val19 of the natural peptide were replaced by phenylalanyl and tyrosyl residues: [Phe13, Tyr19]-MCH. The peptide was synthesized by the continuous-flow solid-phase methodology using Fmoc-strategy and polyhipe PA 500 and PEG-PS resins. The linear MCH peptides with either acetamidomethyl-protected or free cysteinyl residues were purified to homogeneity and cyclized by iodine oxidation, yielding the final product with the correct molecular weight of 2434.61. Radioiodination of the C-terminal tyrosine was carried out enzymatically using solid-phase bound glucose oxidase/lactoperoxidase, followed by purification on a reversed-phase mini-column and by high-pressure liquid chromatography. The resulting [125I]-[Phe13, Tyr19]-MCH tracer was the first radiolabelled MCH peptide suitable for radioreceptor assay: saturation binding analysis using mouse G4F-7 melanoma cells demonstrated the presence of 1090 MCH receptors per cell. The dissociation constant (KD) was 1.18 x 10(-10) M, indicating high-affinity MCH receptors on these cells. MCH receptors were also found in other cell lines such as mouse B16-F1 and G4F and human RE melanoma cells as well as in PC12 and COS-7 cells. Competition binding analyses with a number of other peptides such as alpha-MSH, neuropeptide Y, substance P and pituitary adenylate cyclase activating peptide, demonstrated that the binding to the MCH receptor is specific. Atrial natriuretic factor was found to be a weak competitor of MCH, indicating topological similarities between MCH and ANF when interacting with MCH receptors.

Synthesis and Biological Evaluation in Vitro of a Selective, High Potency Peptide Agonist of Human Melanin-concentrating Hormone Action at Human Melanin-concentrating Hormone Receptor 1

“…Tables I-III were prepared by solid-phase syntheses as described under "Experimental Procedures." They were evaluated for their respective binding affinities for cloned human MCH receptor 1 and 2 in the competition binding assays with 125 I-[Phe 13 ,Tyr 19 ]hMCH as the radiolabeled ligand (28) and, also, for their ability to stimulate inositol trisphosphate-coupled mobilization of intracellular calcium in human HEK-293 cells expressing hMCH-1R and hMCH-2R (24,29,30).…”

Structure-Activity Relationship Studies of Melanin-concentrating Hormone (MCH)-related Peptide Ligands at SLC-1, the Human MCH Receptor

“…First strand cDNA (corresponding to 1 g of total RNA) was subjected to 35 cycles of amplification using the forward primer 5Ј-GAGACCCAAGCTTCTGGATGGACCTGGAAGCCT-3Ј and the reverse primer 5Ј-GATGACGCGGCCGCTCAGGTGCCTTTGCTTTCTG-3Ј (33). After an initial cycle of denaturation at 94°C for 1 min, polymerase chain reaction was carried out for 35 cycles with the following cycle conditions: 94°C, 1 min; 55°C, 1 min; 72°C, 3 min with a postincubation of 72°C for 7 min.…”

“…It was also shown that the MCH ring was essential (32) and that any modification (including amino acid deletion) of this ring was deleterious for biological activity (30). A second series of data have been published more recently using membranes from mammalian cells that were tested for their capacity to bind the current labeled derivative of MCH, [Phe 13 ,Tyr 19 ]MCH (15,16,(33)(34)(35)(36). Among other discrepancies, the reported affinities for salmon MCH in the cell lines used (16,34) were quite different from those reported at the rat or human cloned receptor (17,21).…”

Identification and characterization of a melanin-concentrating hormone receptor

“…3C). [Phe 13 , Tyr 19 ]-MCH, a synthetic analog of MCH known to bind to and activate MCHR1 (8,12,22), also activates MCHR2, with a slightly lower potency compared with MCH. Other peptides from the same MCH precursor neuropeptide-EI and neuropeptide GE (23,24) were inactive for MCHR2.…”