Cloning and characterization of a PAC1 receptor hop-1 splice variant in goldfish (Carassius auratus)

Abstract

In several vertebrates, it has been demonstrated that alternative splicing of PAC1 receptor (PAC1-R) transcripts can generate a number of functional receptor variants which utilize different signal transduction pathways to mediate their activities. As PACAP is a physiological growth hormone-releasing factor in fish, and PACAP and the PAC1-R are highly conserved in vertebrate evolution, it would be of interest to investigate the structure and cellular distribution, particularly in the pituitary, of PAC1-R splice variants in a fish model. Our laboratory has previously cloned a receptor cDNA corresponding to the goldfish PAC1-R-s (goldfish PAC1-R-short). In the present study, a goldfish PAC1-R-hop1 variant was characterized. Functional expression of goldfish PAC1-R-s and PAC1-R-hop1 in Chinese Hamster Ovary cells revealed that, upon stimulation by ovine PACAP38, these receptor variants exhibited similar EC 50 values (8.7 ± 1.5 and 8.8 ± 1.9 nM, respectively) and maximal responses in activating intracellular cAMP production. The presence and expression levels of these transcripts were measured by quantitative real-time PCR in the brain, heart, pituitary and male gonad, and goldfish PAC1-R-s were found to be the predominant form. In situ hybridization of goldfish PAC1-R in the pituitary revealed its prevalent presence in the pars distalis. In summary, the present study provides information to confirm the role of PACAP in the pituitary and to elucidate the pleiotropic effects of PACAP in fish. © 2005 Elsevier Inc. All rights reserved.