Isolation and cloning of VIP receptors.

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The binding characteristic of vasoactive intestinal peptide (VIP) receptors was examined using radioligand binding assay with iodinated VIP and unlabelled peptides in liver mambranes of the dog. VIP binding to canine liver membranes was rapid, reversible, saturable, specific and dependent on temperature. Guanine nucleotides dose-dependently inhibited VIP binding. Stoichiometric data suggested the presence of two classes of VIP binding sites. The order of potency by competition experiments with unlabelled peptides was: VIP > pituitary adenylate cyclase activating peptide (PACAP)-27 > PACAP-38 >> peptide histidine isoleucine (PHI) = secretin in the dog and PACAP-27 > PACAP-38 > VIP > PHI > secretin in the rat. PHI and secretin were about 5000 times less potent than VIP in the dog but about 100 times less potent in the rat. It was concluded that the VIP receptors in canine liver membranes possesses recognition sites for VIP which were distinct from those in rat liver membranes. To obtain DNA probe for screening. VIP receptor gene of the rat liver was amplified by polymerase chain reaction using synthesized oligonucleotide primers. The products, 540-base-pair fragments, were purified and served as the probe after labelling. A cDNA library (300000 clones) derived from canine liver was screened under low stringency conditions. Six positive colonies were detected. Four out of 6 clones with larger inserts (1-1.5 kbb) were subjected to nucleotide sequence analysis. #35 clone has a nucleic acid sequence identical to parathyroid hormone receptors of the rat and opossum. #102 clone has a nucleic acid sequence similar to rat VIP receptors but not #201 and #513 clones. The analysis of #102 clone is in progress.