The pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1) is a heptahelical, G protein-coupled receptor that has been shown to be expressed by non-squamous lung cancer and breast cancer cell lines, and to be coupled to the growth of these tumors. We have previously shown that PACAP and its receptor, PAC1, are expressed in rat colonic tissue. In this study, we used polyclonal antibodies directed against the COOH terminal of PAC1, as well as fluorescently labeled PACAP, Fluor-PACAP, to demonstrate the expression of PAC1 on HCT8 human colonic tumor cells, using FACS analysis and confocal laser scanning microscopy. Similarly, anti-PACAP polyclonal antibodies were used to confirm the expression of PACAP hormone by this cell line. We then investigated the signal transduction properties of PAC1 in these tumor cells. PACAP-38 elevated intracellular cAMP levels in a dose-dependent manner, with a half-maximal (EC(50)) stimulation of approximately 3 nM. In addition, PACAP-38 stimulation caused an increase in cytosolic Ca(2+) concentration [Ca(2+)](i), which was partially inhibited by the PACAP antagonist, PACAP-(6-38). Finally, we studied the potential role of PACAP upon the growth of these tumor cells. We found that PACAP-38, but not VIP, increased the number of viable HCT8 cells, as measured by MTT activity. We also demonstrated that HCT8 cells expressed the Fas receptor (Fas-R/CD95), which was subsequently down-regulated upon activation with PACAP-38, further suggesting a possible role for PACAP in the growth and survival of these tumor cells. These data indicate that HCT8 human colon tumor cells express PAC1 and produce PACAP hormone. Furthermore, PAC1 activation is coupled to adenylate cyclase, increase cytosolic [Ca(2+)](i), and cellular proliferation. Therefore, PACAP is capable of increasing the number of viable cells and regulating Fas-R expression in a human colonic cancer cell line, suggesting that PACAP might play a role in the regulation of colon cancer growth and modulation of T lymphocyte anti-tumoral response via the Fas-R/Fas-L apoptotic pathway.

The pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 (PAC1) receptor is a G proteincoupled receptor and class II receptor member. The receptor domains critical for signaling are unknown. To explore the role of the C terminus, truncations of 63 residues (Tr406), 53 residues (Tr416), 49 residues (Tr420), 44 residues (Tr424), and 37 residues (Tr433) were constructed and expressed in NIH/3T3 cells, and immunofluorescence, radioligand binding, adenylyl cyclase (AC) and phospholipase C (PLC) assays were performed. 125 It has been previously demonstrated for receptors belonging to the class I heptahelical receptor family that specific intracellular amino acid residues are critical for internalization and coupling to guanine nucleotide-binding proteins (G proteins). These cytoplasmic domains have been partially characterized for the ␣-and ␤-adrenergic receptors (1, 2), the muscarinic receptors (3), and for rhodopsin (4). Studies of the ␤ 2 -adrenergic (2) and muscarinic receptors (3) have suggested that substitution, deletion, and/or insertion of residues in the third intracellular loop affect the ability of these receptors to couple to G proteins.At present, the role of the cytoplasmic domains for members of the class II family for receptor coupling to intracellular G proteins has not been fully explored. Based on studies with receptors belonging to the class I receptor family, we speculated that amino acids in either the third intracellular loop or the C terminus of the pituitary adenylate cyclase-activating polypeptide receptor (PAC1), 1 may be important for coupling to G proteins (5). Furthermore, the C terminus has been proposed to be necessary for the formation of a functional receptor-G protein complex and to result in heterodimerization for some GPCRs such as type B receptor for GABA and GABA B -like orphan receptor (6).Pituitary adenylate cyclase-activating polypeptide (PACAP) hormone was originally isolated from bovine hypothalamus and is one of the most recently identified peptides in the VIP/ secretin/glucagon family (7). The cloning of the rat and human PAC1 cDNA and genes identified a receptor protein composed of ϳ468 amino acids containing seven transmembrane domains that are characteristic of G protein-coupled receptors (5, 8). The PAC1 belongs to the class II GPCR superfamily that includes VPAC1 (the classical VIP1 receptor), VPAC2 (VIP2 receptor), secretin, glucagon, and calcitonin receptors (9); these molecules can be distinguished on the basis of their relative affinities for the agonists PACAP-27, PACAP-38, VIP, and helodermin (10). The cloning of the rat and human PAC1 gene have identified two unique exons that can be alternatively spliced yielding different third intracellular loops. The four major splice variants are named Null, Hip, Hop,8), and they have been shown to be differentially expressed in different tissues and are able to couple with different efficacies and potencies to phospholipase C (PLC) and adenylyl cyclase (AC) (5,8). Therefore, the expression of splice varian...

Fluor-PACAP, a fluorescent derivative of PACAP-27, has been confirmed to share a high affinity for PAC1 receptors transfected into NIH/3T3 cells and to have comparable pharmacological characteristics to the unconjugated, native form. Through competitive binding with 125I-PACAP-27, the two ligands exhibited similar dose- dependent inhibition. Additional examination of the efficacy of activating adenylyl cyclase revealed that both ligands analogously stimulated the production of cyclic AMP. Furthermore, PAC1 internalization visualized by our Fluor-PACAP, is compareable to that performed with the radioligand, 125I-PACAP-27, with maximal internalization achieved within thirty minutes. Thus, Fluor-PACAP exhibits intracellular signaling abilities homologous to the native ligand.