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

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 mu l of exudate y ielded 380 mu g of purified peptide. Mass spectrometric analysis and gas ph ase sequencing of the purified peptide as well as chemical synthesis and cD NA analysis were consistent with the structure SCDTSTCATQRLADFLSRSGGIGSPDFV PTDVSANSF amide and the presence of a disulfide bridge linking Cys(2) and C ys(7), The skin peptide, named skin calcitonin gene-related peptide, differ s significantly from all other members of the calcitonin gene-related pepti de family of peptides at nine positions but binds with high affinity to cal citonin gene-related peptide receptors in the rat brain and acts as an agon ist in the rat vas deferens bioassay with potencies equal to those of human CGRP, Reverse transcriptase-polymerase chain reaction coupled with cDNA cl oning and sequencing demonstrated that skin calcitonin gene-related peptide isolated in the skin is identical to that present in the frog's central an d enteric nervous systems. These data, which indicate for the first time th e existence of calcitonin gene-related peptide in the frog skin, add furthe r 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.