Structural and functional characterization of pituitary adenylyl cyclase-activating polypeptide (PACAP)/PACAP-related peptide (PRP) and its receptor in olive flounder (Paralichthys olivaceus)

摘要

We identified full-length cDNAs encoding pituitary adenylyl cyclase-activating polypeptide (PACAP), PACAP-related peptide (PRP), and PACAP-specific receptor (PAC1R) from olive flounder, Paralichthys olivaceus. Two variant mRNA forms were created by alternative splicing. Comparison of genomic and cDNA sequences of the PRP-PACAP precursor revealed that skipping of exon 4 within PRP resulted in two variant transcripts: a long form encoding both PRP and PACAP and a short formencoding PACAP only. Both transcripts were constitutively observed only in the brain,whereas the short form appeared in gut tissues, such as the intestine and pyloric cecum in fish challenged with a pathogen, but not in healthy fish. Furthermore, expression of the long PRP/PACAP transcript gradually increased in the intestine of flounder challenged with bacteria, suggesting that PRP and/or PACAP may serve as a regulator(s) of the immune system, especially in the gastrointestinal tract of olive flounder. The biological functions of PACAP and PRPwere investigated by exogenous treatment of flounder embryogenic cells (hirame natural embryonic cells, HINAE cells) with synthetic peptides of fPACAP-38 and/or fPRP-45. Intracellular cyclic adenosine monophosphate (cAMP) production in PAC1R-overexpressing HINAE cells was regulated by fPACAP-38 in a concentration-dependent manner, but was not regulated by fPRP-45. Results fromreal-time quantitative polymerase chain reaction revealed that PAC1R mRNA was specifically induced by fPACAP-38 but not by fPRP-45; PACAP significantly increased TNF-α mRNA but not growth hormone (GH) mRNA in HINAE cells; however, PRP affected GH but not TNF-α mRNA expression. These results suggest that the expression ratio of PRP and PACAP is regulated at the transcriptional level depending on the tissues and conditions, and that the unique biological roles of PRP and PACAP differ from that of mammalian PRP.