Molecular cloning and functional expression of the pituitary adenylate cyclase-activating polypeptide type I receptor.

Abstract

Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide belong to the vasoactive intestinal peptide (VIP)/secretin/glucagon family of peptides, Interacts with a distinct hig-afflnity receptor (type I receptor) on a number of tissues. These PACAP type I receptors have a hh affinity for PACAP and a low affhity for VIP and are present in the hypothalamus and anterior pituiltary, where they regulate the release of adrenocorticotropin, luteinizing hormone, growth hormone, and prolactin, and in the adrenal medulla, where they regulate the release of epinephrine. Type I PACAP receptors are also present in high concentrations in tticular germ ceils, where they may re spermatogenesis, and some trnsformed cefl lines, such as the rat pancreatic acinar carinoma cel AR4-2J. Here we report the moleular cloin and functional expression ofthe PACAP type I receptor isolated from an AR4-2J cell cDNA library by cross-hybridization screening with a rat VIP receptor cDNA. (4); and in the gastrointestinal tract, where they cause smooth muscle relaxation and secretion (11)(12)(13). Type I receptors have also been described on cultured rat astrocyes (14), a rat adrenal pheochromocytoma cell line, PC12H (9), a rat pancreatic acinar carcinoma cell line, AR4-2J (15), and a human neuroblastoma cell line, .Affinity crosslinking studies of type I PACAP receptors on bovine brain membranes demonstrate a 57-kDa molecular species that is modulated in its affinity for PACAP by guanine nucleotides (17) and suggest its membership in the guanine nucleotide-binding regulatory protein (G protein)-coupled family ofreceptors similar to the recently cloned VIP (18) and secretin receptors (19).Further knowledge of the molecular structure of the PACAP type I receptor and its gene would enhance our understanding of its distribution, function, and regulation. We describe here the identification of cDNA clones resulting from cross-hybridization screening of a cDNA library constructed from the rat pancreatic carcinoma cell line AR4-2J with a rat VIP receptor cDNA probe.t These clones encode a protein that has an affinity for PACAP-38, PACAP-27, and VIP consistent with the type I PACAP receptor pharmacology, has a high degree of homology to both the VIP and secretin receptors, and mediates PACAP-stimulated accumulation of intracellular cAMP. MATERIALS AND METHODScDNA Library Construction and Isolation of cDNA Clones. A cDNA library was constructed from AR4-2J cells as described previously (20). The library (%7 x 105 plaques) was screened with a 32P-labeled, randomly primed probe (21) corresponding to the complete coding region of the rat VIP receptor cDNA (18) that was PCR cloned from rat pancreatic cDNA. The library was initially screened under conditions of low and later high stringency [three 20-min washes at 37°C with 2x SSC/0.1% SDS for low-stringency screening and three 20-min washes at 420C with 0.1x SSC/0.1% SDS for high-stringency screening (lx SSC = 150 mM NaCl/15 mM sodium citrate, pH 7.0)] (22). Several clones that hybridi...

Direct cAMP Signaling through G-Protein-Coupled Receptors Mediates Growth Cone Attraction Induced by Pituitary Adenylate Cyclase-Activating Polypeptide

“…In particular, PAC1 splice variants activate a number of signaling cascades, including the activation of adenylate cyclase, phosphatidyl inositol turnover, and L-type Ca 2ϩ channels (Pisegna and Wank, 1993;Spengler et al, 1993;Chatterjee et al, 1996). For instance, PAC1 hop splice variant activation increases PI turnover, protein kinase C localization, and intracellular Ca 2ϩ mobilization, whereas PAC1 short activation only stimulates adenylate cyclase (Lu et al, 1998;DiCicco-Bloom et al, 2000;Nicot and DiCicco-Bloom, 2001).…”

“…Different splice variants of PAC1 receptors elicit different intracellular signaling cascades besides the cAMP pathway, including PLC, PI-3 kinases, and L-type Ca 2ϩ channels (Pisegna and Wank, 1993;Spengler et al, 1993;DiCicco-Bloom et al, 2000;Nicot and DiCicco-Bloom, 2001). To determine whether PACAP elicits Ca 2ϩ responses in cultured Xenopus spinal neurons, we used fura-2 ratiometric imaging to measure intracellular Ca 2ϩ concentrations ([Ca 2ϩ ] i ).…”

Functional Characterization of Structural Alterations in the Sequence of the Vasodilatory Peptide Maxadilan Yields a Pituitary Adenylate Cyclase-activating Peptide Type 1 Receptor-specific Antagonist

“…Like other neuropeptides, PACAP possesses various physiological functions, including neurotransmission, vasodilatory actions, and endocrinological effects. Cloning of the PACAP receptors reveals three distinct subtypes (2)(3)(4). Two of the three receptors also function as receptors for VIP.…”

A Natural Variant Type II G Protein-coupled Receptor for Vasoactive Intestinal Peptide with Altered Function

“…To determine the extent of VIP suppression of generation of human IL-2 by stimulated Jurkat T cells, replicate 0.6-ml aliquots of 2 ϫ 10 6 Jurkat cells/ml of complete RPMI 1640 were added to 24-well plates that had been precoated with 0.75 g/well of mouse anti-human CD3 monoclonal antibody (PharMingen). After addition of 10 Ϫ9 to 10 Ϫ6 M VIP or 10 Ϫ6 M VPAC 1 -selective agonist or VPAC 2 -selective agonist, wells received 20 ng/ml phorbol myristate acetate, and incubations were continued for 48 h at 37°C in 5% CO 2 in humidified air.…”