CDB15:0000026 ADCYAP1 — ADCYAP1R1

ABSTRACT: Three full-length cDNAs encoding functional splice variants of the pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1) were isolated from Y-79 retinoblastoma cells and human cerebellum. Although the third intracellular loops of the three splice variants were identical, their N-terminal extracellular domains differed. The first full-length PAC1 variant, PAC1normal (PAC1n), encoded the entire N-terminus, whereas the second variant named PAC1short (PAC1s) was deleted by 21 amino acids (residues 89-109). Finally, the third variant, named PAC1very short (PAC1vs), was deleted by 57 amino acids (residues 53-109). Using semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis, it was established that all three variants were expressed in neuronal tissues. Binding- and cAMP studies using human embryonic kidney 293 (HEK293) cells stably transfected with PAC1n, PAC1s and PAC1vs showed significant differences in the affinities and selectivities towards PACAP38, PACAP27 and VIP. PAC1n bound PACAP38 and PACAP27 with affinities in the low nanomolar range whereas VIP was bound with up to 400-fold lower affinity. PAC1vs preferentially bound PACAP38 (Ki=121 nM) and PACAP27 (Ki=129 nM) over VIP (Ki>1000 nM) but with 100-fold lower affinity than PAC1n. Surprisingly, PAC1s unselectively bound all three ligands with high affinity. These data indicate that residues 53-88 within the N-terminal domain of the PAC1 are important for high affinity ligand binding, whereas residues 89-109 determine the receptor's ligand selectivity.

ABSTRACT: The effects of a (N-stearyl, Norleucine17) vasoactive intestinal peptide hybrid ((SN)VIPhybrid) on cells stably transfected with VPAC,, VPAC2, or PAC1 receptors were investigated. (SN)VIPhybrid inhibited specific 125I-VIP binding to membranes derived from CHO cells transfected with VPAC, or VPAC2 receptors with high affinity (IC50 = 30 and 50 nM). (SN)VIPhyb inhibited specific 125I-PACAP-27 binding to membranes derived from NIH/3T3 cells transfected with PAC1 receptors with high affinity (IC50 = 65 nM). PACAP-27 caused cAMP elevation in NIH/3T3 cells transfected with PAC1 receptors and the increase cAMP caused by pituitary adenylated cyclase (PACAP) was inhibited by (SN)VIPhyb. Also, the increase in cAMP caused by VIP using CHO cells transfected with VPAC1 or VPAC2 receptors was antagonized by (SN)VIPhyb. These results indicate that (SN)VIPhyb is an antagonist for VPAC1, VPAC2, and PAC1 receptors.

ABSTRACT: Previous studies have shown that human fetal adrenal gland from 17- to 20-week-old fetuses expressed pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, which were localized on chromaffin cells. The aim of the present study was to identify PACAP receptor isoforms and to determine whether PACAP can affect intracellular calcium concentration ([Ca(2+)](i)) and catecholamine secretion. Using primary cultures and specific stimulation of chromaffin cells, we demonstrate that PACAP-38 induced an increase in [Ca(2+)](i) that was blocked by PACAP (6-38), was independent of external Ca(2+), and originated from thapsigargin-insensitive internal stores. The PACAP-triggered Ca(2+) increase was not affected by inhibition of PLC beta (preincubation with U-73122) or by pretreatment of cells with Xestospongin C, indicating that the inositol 1,4,5-triphosphate-sensitive stores were not mobilized. However, forskolin (FSK), which raises cytosolic cAMP, induced an increase in Ca(2+) similar to that recorded with PACAP-38. Blockage of PKA by H-89 or (R(p))-cAMPS suppressed both PACAP-38 and FSK calcium responses. The effect of PACAP-38 was also abolished by emptying the caffeine/ryanodine-sensitive Ca(2+) stores. Furthermore, treatment of cells with orthovanadate (100 microm) impaired Ca(2+) reloading of PACAP-sensitive stores indicating that PACAP-38 can mobilize Ca(2+) from secretory vesicles. Moreover, PACAP induced catecholamine secretion by chromaffin cells. It is concluded that PACAP-38, through the PAC(1) receptor, acts as a neurotransmitter in human fetal chromaffin cells inducing catecholamine secretion, through nonclassical, recently described, ryanodine/caffeine-sensitive pools, involving a cAMP- and PKA-dependent phosphorylation mechanism.

ABSTRACT: VPAC/PAC receptor activation classically results in cyclic-AMP production, with limited reports evaluating calcium signalling. These studies systematically characterise intracellular cyclic-AMP ([cAMP](i)) and calcium ([Ca(2+)](i)) responses in CHO-cells expressing recombinant human (h) VPAC/PAC receptors (hVPAC(1)R, hVPAC(2)R, hPAC(1)R), using two simple, non-radioactive, HT-amenable assays. The rank order of potency (ROP) of the agonists VIP, PACAP-27 and PACAP-38 was similar in both assays for each individual receptor subtype, although potencies (EC(50)) in the [Ca(2+)](i) assay were approximately 100-fold lower. Importantly, this shift was also evident in SHSY-5Y cells endogenously expressing hPAC(1)R. Furthermore, [Ala(11,22,28)]VIP and maxadilan were selective hVPAC(1)R and hPAC(1)R agonists, respectively, and although R3P65 had no demonstrable hVPAC(2)R selectivity, these compounds exhibited comparable reductions in [Ca(2+)](i) EC(50) values. In contrast, PG97-269 and PG99-465, putatively selective hVPAC(1)R and hVPAC(2)R antagonists, respectively, were marginally less potent in [cAMP](i) studies, whereas M65 was equipotent at hPAC(1)R. Moreover, PG99-465 alone increased [cAMP](i) at all three hVPAC/PAC receptor subtypes, with full hVPAC(1)R and hPAC(1)R agonism. With equivalent agonist ROPs generated in both assays, [Ca(2+)](i) signalling provides an alternative approach to examine hVPAC/PAC receptor pharmacology. However, these studies underscore the paucity of receptor selective compounds, complexities in comparing drug potencies across assays, and the pleiotropic nature of VPAC/PAC-receptor signalling.

ABSTRACT: Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide belonging to the vasoactive intestinal polypeptide/glucagon/secretin family. It is widely distributed in the body, and a variety of biological actions have been reported. PACAP exerts its biological effects by binding to specific receptors that are coupled to GTP-binding proteins. Recent studies have shown that there is a family of PACAP receptors (PACAPRs), and two members of this family have been identified. We report here the cloning, functional expression, and tissue distribution of a third PACAPR subtype, designated PACAPR-3. The cDNA encoding PACAPR-3 has been isolated from a mouse insulin-secreting beta-cell line MIN6 cDNA library. Mouse PACAPR-3 is a protein of 437 amino acids that has 50% and 51% identity with rat PACAP type I and type II receptors, respectively. Expression of recombinant mouse PACAPR-3 in mammalian cells shows that it binds to vasoactive intestinal polypeptide as well as PACAP-38 and -27, with a slightly higher affinity for PACAP-38, and is positively coupled to adenylate cyclase. The expression of PACAPR-3 in Xenopus oocytes indicates that calcium-activated chloride currents are evoked by PACAP and vasoactive intestinal polypeptide, suggesting that PACAPR-3 can also be coupled to phospholipase C. RNA blot analysis studies reveal that PACAPR-3 mRNA is expressed at high levels in MIN6, at moderate levels in pancreatic islets and other insulin-secreting cell lines, HIT-T15 and RINm5F, as well as in the lung, brain, stomach, and colon, and at low levels in the heart. Furthermore, insulin secretion from MIN6 cells is significantly stimulated by PACAP-38. These results suggest that the diverse biological effects of PACAP are mediated by a family of structurally related proteins and that PACAPR-3 participates in the regulation of insulin secretion.

ABSTRACT: Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38-amino acid peptide (PACAP38) or a truncated peptide with the same 27 amino-terminal residues (PACAP27). The PACAP receptor was solubilized from bovine brain membranes with digitonin and purified 30-fold by the combination of DEAE-Toyopearl and hydroxylapatite chromatographic analyses. The partially purified PACAP receptors were mixed with biotinylated PACAP27 to form receptor-ligand complexes and then adsorbed onto avidin-agarose. The adsorbed PACAP receptors were eluted with an acidic buffer containing 1.0 M NaCl (pH 4.0). The eluted receptors were purified further by hydroxylapatite and gel filtration chromatography. A single protein band with a M(r) = 55,000-60,000 was found in the final preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. Affinity labeling of the purified receptors with 125I-PACAP27 labeled the M(r) 55,000-60,000 protein specifically. The dissociation constant and the specific activity of the purified receptors were 25.8 pM and 17.2 nmol of ligand binding per mg of protein, respectively. Inhibitory constants determined by competitive binding experiments were 30.0 pM for PACAP27, 4.6 pM for PACAP38, and 37.3 nM for vasoactive intestinal peptide. Therefore, the purified PACAP receptor retained high affinity and ligand specificity. The sequence of the amino-terminal 29 residues was derived from the purified receptor.

ABSTRACT: Pituitary adenylate cyclase-activating polypeptide (PACAP)-27 and PACAP-38 are neuropeptides of the vasoactive intestinal peptide/secretin/glucagon family. We previously described alternative splicing of the region encoding the third intracellular loop of the PACAP receptor generating six isoforms with differential signal transduction properties (Spengler, D., Waeber, C., Pantaloni, C., Holsboer, F., Bockaert, J., Seeburg, P. H., and Journot, L. (1993) Nature 365, 170-175). In addition, we demonstrated that the potencies of the two forms of PACAP are similar for adenylate cyclase stimulation, whereas PACAP-38 is more potent than PACAP-27 in phospholipase C activation. In the present work, we document the existence of a new splice variant of the PACAP receptor that was characterized by a 21-amino-acid deletion in the N-terminal extracellular domain. We demonstrate that this domain modulates receptor selectivity with respect to PACAP-27 and -38 binding and controls the relative potencies of the two agonists in phospholipase C stimulation.