Essential structural motif in the C-terminus of the PACAP type I receptor for signal transduction and internalization.
The objectives of the study reported here were to identify amino acid residues of the C-terminus that are critical for intracellular signaling. A total of nine amino acid substitution and truncation mutants were constructed by PCR and confirmed by sequencing. Mutant and wildtype receptors were stably transfected into NIH/3T3 fibroblasts and studied for their ability to bind PACAP-27 and activate phospholipase C (PLC) and adenylyl cyclase (AC). Receptor affinity of 125I-PACAP-27 for the wildtype and mutants were similar (Kd = 0.6-1.5 nM). However, truncation of the entire 63 amino acids of the hPAC1 resulted in no signaling to either AC or IP. Addition of the proximal 10 amino acids of the C-terminus failed to restore AC or IP signaling, whereas addition of the proximal 27 amino acids of the C-terminus resulted in reconstitution of complete AC and IP responses, identical to the WT. Point mutations within this 17 amino acid region identified specific amino acids involved in PAC1 signaling. These results indicate that a structural motif within the proximal region of the carboxyl terminus is critical for G protein coupling.
Pisegna
J R
JR
Division of Gastroenterology and Hepatology, VA Greater Los Angeles Healthcare System. jpisegna@ucla.edu
Lyu
R M
RM
Germano
P M
PM
D016475:3T3 Cells; D000262:Adenylyl Cyclases / Q000378:metabolism; D020816:Amino Acid Motifs; D000818:Animals; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007295:Inositol Phosphates / Q000378:metabolism; D051379:Mice; D009479:Neuropeptides / Q000378:metabolism; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D017354:Point Mutation; 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*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D015398:Signal Transduction; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism
C495401:ADCYAP1 protein, human; C495503:ADCYAP1R1 protein, human; C495402:Adcyap1 protein, mouse; C495504:Adcyap1r1 protein, mouse; D007295:Inositol Phosphates; D009479:Neuropeptides; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; D011979:Receptors, Pituitary Hormone; D011994:Recombinant Proteins; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases