Considerable plasticity can occur within the amino acid sequence of amphiphilic peptide hormones. This is particularly evident within the corticotropin-releasing factor (CRF) family of peptides where, despite less than 15% sequence similarity among the four paralogous lineages, all are capable of acting as high affinity ligands to members of the CRF receptor family. This suggests that these peptides could undergo many mutational changes and remain as high affinity ligands to their receptors as long as the functional motifs do not change radically. Because paralogous peptide lineages are a product of genome duplications, additional genes encoding peptide-like sequences, which through mutation have lost their functional integrity, may exist. Function to these sequences may be restored if the appropriate motifs are reinserted into the primary structure. We screened rat genomic DNA with highly degenerate polymerase chain reaction (PCR) primers targeted to hybridize with the termini of CRF-related sequences. One set of sauvagine-based primers hybridized with a 120-bp sequence. The theoretical peptide sequence (SV4) showed similarity to the CRF family of peptides at the primary structure level. The encoded sequence was prepared by solid-phase synthesis and its activity assayed against mouse R1 and human R1/R2 receptors. SV4 did not bind to either mouse or human variants of the R1 receptor, but did bind to the R2 receptor with an affinity comparable to human CRF. SV4 exhibited a similar efficacy of cellular activation as CRF in trials quantifying the acidification rate of human R2alpha-transfected Chinese hamster ovary (CHO) cells, but not R1-transfected cells. SV4 utilizes adenylate cyclase as the principal secondary messenger of R2 signal transduction but, unlike urocortin or sauvagine, does not activate guanylate cyclase-, calcium- or mitogen-activated protein (MAP) kinase-mediated pathways. These data suggest that this artificial peptide may be useful to understand the cyclic adenosine monophosphate (cAMP)-dependent component of the CRF-R2 signal transduction cascade, and that additional sequences in the genome may be used to engineer bioactive peptides.