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. 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)I-PACAP-27 binding (K(d) = 0.6-1.5 nm) for the Tr406 and Tr433 were similar to wild type Hop and Null splice variants (K(d) = approximately 1.1 nm). Although internalization of ligand for both the Tr406 and Tr433 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. Despite similar binding properties Tr406 and Tr416 mutants showed no AC or PLC response. Addition of 14 amino acids distal to HopTr406 resulted in normal AC and PLC responses. Site-directed mutagenesis indicated that Arg(416) and Ser(417) are essential for G protein activation. The proximal C terminus mediates signal transduction, and the distal is involved with internalization. Two residues within the C terminus, Arg(416) and Ser(417) conserved among class II receptors are the likely sites for G protein coupling.
Lyu
R M
RM
CURE, Veterans Affairs/UCLA Digestive Diseases Research Center, Veterans Affairs Greater Los Angeles Health Care System and the Department of Medicine, UCLA, Los Angeles, California 90073, USA.
Germano
P M
PM
Choi
J K
JK
Le
S V
SV
Pisegna
J R
JR
D016475:3T3 Cells; D000262:Adenylyl Cyclases / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D000242:Cyclic AMP / Q000378:metabolism; D015536:Down-Regulation; D004705:Endocytosis; D005455:Fluorescent Antibody Technique; D006207:Half-Life; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D007457:Iodine Radioisotopes; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010902:Pituitary Gland / Q000201:enzymology / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; D011979:Receptors, Pituitary Hormone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D015398:Signal Transduction; D014162:Transfection
C495503:ADCYAP1R1 protein, human; C495504:Adcyap1r1 protein, mouse; D007295:Inositol Phosphates; D007457:Iodine Radioisotopes; D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; D011979:Receptors, Pituitary Hormone; D000242:Cyclic AMP; D000262:Adenylyl Cyclases