The functional recombinant first extracellular (EC1) domain of PACAP receptor PAC1 normal form (PAC1-EC1(N)) recognizes selective ligands and stimulates the proliferation of PAC1-CHO cells.
PAC1 is a pituitary adenylate cyclase-activating polypeptide (PACAP) preferring receptor, which is abundant in the central and peripheral nervous systems. PAC1 belongs to the class B family of G protein-coupled receptors (GPCRs). The N-terminal first extracellular (EC1) domain of PAC1 is responsible for ligand recognition and binding. In this study, the recombinant EC1 domain of the PAC1 normal (N) form (amino acids 21-155) with 6His tag at the C-terminus (named PAC1-EC1(N)) was first expressed in an Escherichia coli strain and purified by an Ni-NTA affinity column. About 6-8mg of recombinant PAC1-EC1(N) protein with purity above 95% was produced from 1L of bacterial culture. Mass spectrum and western blot were used to identify the recombinant PAC1-EC1(N). Intrinsic tryptophan fluorescence (ITF) assays showed that the purified PAC1-EC1(N) protein was able to recognize and bind to the PAC1 selective agonist maxadilan, the antagonist M65 and vasoactive intestinal polypeptide (VIP). Maxadilan and M65 had higher affinities for PAC1-EC1(N) than VIP. The results of MTT assays showed that PAC1-EC1(N) stimulated the viability of PAC-CHO cells but blocked the effects of maxadilan on the proliferation of CHO cells expressing PAC1 (PAC1-CHO), indicating that the functional soluble PAC1-EC1(N) may act as a regulator for the activation of PAC1.
Yu
Rongjie
R
Bio-engineering Institute of Jinan University, Jinan University, 510632 China. rongjie yu1123@163.com
Li
Juan
J
Wang
Jingjing
J
Liu
Xiaofei
X
Huang
Lin
L
Ding
Yong
Y
Chen
Jiansu
J
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D015740:Calcitonin Gene-Related Peptide / Q000378:metabolism / Q000494:pharmacology; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011994:Recombinant Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism; D017384:Sequence Deletion; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism / Q000494:pharmacology
D019476:Insect Proteins; D008024:Ligands; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; D011994:Recombinant Proteins; C069147:maxadilan protein, insect; D014660:Vasoactive Intestinal Peptide; D015740:Calcitonin Gene-Related Peptide