Characterization of the pharmacology, signal transduction and internalization of the fluorescent PACAP ligand, fluor-PACAP, on NIH/3T3 cells expressing PAC1.
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.
Germano
P M
PM
CURE: VA/UCLA Digestive Diseases Research Center, VA Greater Los Angeles Health Care System and Dept of Medicine, the University of California, Los Angeles, Los Angeles, CA 90073, USA.
Stalter
J
J
Le
S V
SV
Wu
M
M
Yamaguchi
D J
DJ
Scott
D
D
Pisegna
J R
JR
D016475:3T3 Cells; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D000242:Cyclic AMP / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes / Q000494:pharmacology; D006834:Hydrazines / Q000494:pharmacology; D007150:Immunohistochemistry; D008024:Ligands; D051379:Mice; D018613:Microscopy, Confocal; D009479:Neuropeptides / Q000737:chemistry* / Q000494:pharmacology; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D011485:Protein Binding; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection
C495402:Adcyap1 protein, mouse; C417561:Alexa 488 hydrazide; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D006834:Hydrazines; D008024:Ligands; D009479:Neuropeptides; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D000242:Cyclic AMP; D000262:Adenylyl Cyclases