Identification of an essential amino acid motif within the C terminus of the pituitary adenylate cyclase-activating polypeptide type I receptor that is critical for signal transduction but not for receptor internalization

The pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 (PAC1 ) receptor is a G protein-coupled receptor and class II receptor member. Th e receptor domains critical for signaling are unknown. To explore the role of the C terminus, truncations of 63 residues(Tr-406), 53 residues (Tr-416) , 49 residues (Tr-420), 44 residues (Tr-424), and 37 residues (Tr-433) were constructed and expressed in NIH/3T3 cells, and immunofluorescence, radiol igand binding, adenylyl cyclase (AC) and phospholipase C (PLC) assays were performed. I-125-PACAP-27 binding (K-d = 0.6-1.5 nM) for the Tr-406 and Tr- 433 were similar to wild type Hop and Null splice variants (K-d = similar t o1.1 nM), Although internalization of ligand for both the Tr-406 and Tr-433 mutants was reduced to 50-60% at 60 min compared with 76-87% for WT, loss of G-protein coupling did not account for differences in internalization. D espite similar binding properties Tr-406 and Tr-416 mutants showed no AC or PLC response. Addition of 14 amino acids distal to HopTr(406) resulted in normal AC and PLC responses. Site-directed mutagenesis indicated that Arg(4 16) and Ser(417) are essential for G protein activation. The proximal C ter minus mediates signal transduction, and the distal is involved with interna lization. Two residues within the C terminus, Arg(416) and Ser(417) conserv ed among class II receptors are the likely sites for G protein coupling.