Shark rectal gland vasoactive intestinal peptide receptor: cloning, functional expression, and regulation of CFTR chloride channels

Abstract

Vasoactive intestinal peptide (VIP) is a secretagogue that mediates chloride secretion in intestinal epithelia. We determined the relative potency of VIP and related peptides in the rectal gland of the elasmobranch dogfish shark and cloned and expressed the VIP receptor (sVIP-R) from this species. In the perfused rectal gland, VIP (5 nM) stimulated chloride secretion from 250 +/- 66 to 2,604 +/- 286 microeq x h(-1) x g(-1); the relative potency of peptide agonists was VIP > PHI = GHRH > PACAP > secretin, where PHI is peptide histidine isoleucine amide, GHRH is growth hormone-releasing hormone, and PACAP is pituitary adenylate cylase activating peptide. The cloned sVIP-R from shark rectal gland (SRG) is only 61% identical to the human VIP-R1. It maintains a long, extracellular NH2 terminus with seven cysteine residues, and has three N-glycosylation sites and eight other residues implicated in VIP binding. Two amino acids considered important for peptide binding in mammals are not present in the shark orthologue. When sVIP-R and the CFTR chloride channel were coexpressed in Xenopus oocytes, VIP increased chloride conductance from 11.3 +/- 2 to 127 +/- 34 microS. The agonist affinity for activating chloride conductance by the cloned receptor was VIP > GHRH = PHI > PACAP > secretin, a profile mirroring that in the perfused gland. The receptor differs from previously cloned VIP-Rs in having a low affinity for PACAP. Expression of both sVIP-R and CFTR mRNA was detected by quantitative PCR in shark rectal gland, intestine, and brain. These studies characterize a unique G protein-coupled receptor from the shark rectal gland that is the oldest cloned VIP-R.

RNA expression in a cartilaginous fish cell line reveals ancient 3′ noncoding regions highly conserved in vertebrates

“…However, comparative approaches using these organisms in cell biology and genomics have been limited by availability of in vitro models of cell culture and a lack of genomic data. Nevertheless, the use of these fish is beginning to contribute important observations in the areas of protein and genome evolution and function (8)(9)(10)(11)(12). The conserved untranslated sequences identified by gene expression analysis of an elasmobranch cell line have been maintained in vertebrates for Ͼ400 million years of evolution and likely reflect retention of function.…”

Divergent CFTR orthologs respond differently to the channel inhibitors CFTRinh-172, glibenclamide, and GlyH-101

“…10-03). Glands were removed from the shark, cannulated, and perfused with elasmobranch Ringer's solution for 30 min to reach basal levels of chloride secretion (ϳ250 eq·h Ϫ1 ·g Ϫ1 ) as previously described (2,5,28,44). At 30 min, forskolin (1 M) and IBMX (100 M) were added to the perfusate to achieve maximal rates of chloride secretion measured at 1-min intervals.…”

“…Ovarian lobules of mature female Xenopus laevis (Xenopus I, Dexter, MI) were obtained, and mature stage V and VI oocytes were selected for two-electrode voltage clamping as described previously (5,63). After 12 h the oocytes were injected with 10 ng cRNA/50 nl or an equivalent volume of water and then stored for 1-2 days in modified Barth solution at 18°C as described previously (5).…”

Pituitary Adenylate Cyclase-Activating Polypeptide and Its Receptors: 20 Years after the Discovery

“…VPAC1-R cDNAs have been cloned in the goldfish (Chow et al, 1997), the fugu (2 isoforms A and B: Cardoso et al, 2004), the dogfish Squalus acanthias (Bewley et al, 2006), the zebrafish (Wu et al, 2008) and the frog R. ridibunda (Alexandre et al, 1999). In contrast to mammals, in which VPAC1-R interacts with VIP and PACAP with similar affinities, VPAC1-R from goldfish and dogfish show higher affinities toward VIP than PACAP.…”