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J Biol Chem, Vol. 275, Issue 8, 5934-5940, February 25, 2000

Isolation, Structure, Synthesis, and Activity of a New Member of the Calcitonin Gene-related Peptide Family from Frog Skin and Molecular Cloning of Its Precursor^*

Aurelia Anne Seon^§, Thierry Nicolas Pierre^§, Virginie Redeker^¶, Claire Lacombe, Antoine Delfour, Pierre Nicolas, and Mohamed Amiche

From the  Laboratoire de Bioactivation des Peptides, Institut Jacques Monod, 2 Place Jussieu, 75251 Paris Cedex 05, France and ^¶ Laboratoire de Neurobiologie, Ecole Supérieure de Physique et de Chimie Industrielles de la Ville de Paris, CNRS UMR 7637, 10 rue Vauquelin, 75005 Paris, France

Calcitonin gene-related peptide has been extracted from the skin exudate of a single living specimen of the frog Phyllomedusa bicolor and purified to homogeneity by a two-step protocol. A total volume of 250 µl of exudate yielded 380 µg of purified peptide. Mass spectrometric analysis and gas phase sequencing of the purified peptide as well as chemical synthesis and cDNA analysis were consistent with the structure SCDTSTCATQRLADFLSRSGGIGSPDFVPTDVSANSF amide and the presence of a disulfide bridge linking Cys² and Cys⁷. The skin peptide, named skin calcitonin gene-related peptide, differs significantly from all other members of the calcitonin gene-related peptide family of peptides at nine positions but binds with high affinity to calcitonin gene-related peptide receptors in the rat brain and acts as an agonist in the rat vas deferens bioassay with potencies equal to those of human CGRP. Reverse transcriptase-polymerase chain reaction coupled with cDNA cloning and sequencing demonstrated that skin calcitonin gene-related peptide isolated in the skin is identical to that present in the frog's central and enteric nervous systems. These data, which indicate for the first time the existence of calcitonin gene-related peptide in the frog skin, add further support to the brain-skin-gut triangle hypothesis as a useful tool in the identification and/or isolation of mammalian peptides that are present in the brain and other tissues in only minute quantities.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) Y18495 (for prepro-skin calcitonin gene-related peptide) and P81564 (for skin calcitonin gene-related peptide).

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