No abstract

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...