Preparation And Identification Of N-Terminal Extracellular Domain Recombinant Proteins Of Vasoactive Intestinal Peptide Receptors VPAC1-R And VPAC2-R And Exploration Of Their Application In Vitro Binding

Objective:The biological functions of pituitary adenylate cyclase activating polypeptide?PACAP?are mediated by three class B G protein-coupled receptors?GPCRs?:VPAC1-R,VPAC2-R and PACA1-R.VPAC1-R and VPAC2-R are shared receptors of vasoactive intestinal peptide?VIP?and PACAP,and PAC1-R is PACAP preferring receptor.N-terminal extracellular domain?EC1?of VPAC1-R,VPAC2-R and PAC1-R plays an important role in ligand recognition and binding,which will be a tool for in vitro screening of agonists and small molecular regulators.In this paper,the recombinant N-terminal extracellular domain of VPAC1-R and VPAC2-R?VPAC1-EC1 and VPAC2-EC1?were prepared using genetic engineering techniques.The in vitro bindings of VPAC1-EC1/VPAC2-EC1 with doxycycline?DOX?/minocycline?MINO?/polypeptide PACAP?28-38?/transmembrane peptide TAT was explored using isothermal titration calorimetry?ITC?assay;we also detected the in vitro binding of a small molecule allosteric regulator named SPAM1 with the N-terminal extracellular domains of PACAP three receptors.The in vitro binding assay based on the preparation of GPCRs.N-terminal extracellular domain will offer a screening tool for new agonists or regulators.Methods:Two genes encoding VPAC1-EC1 and VPAC2-EC1 were synthesized by codon optimization and labeling with 6×His purification tag.The engineering bacteria pET30a-6His-VPAC1-EC1-BL21?DE3?and pET30a-VPAC2-EC1-BL21?DE3?were constructed.After the fermentation conditions were explored,two recombinant proteins VPAC1-EC1 and VPAC2-EC1 were prepared by inclusion body denaturation and purification with Ni-NTA affinity chromatography column.After Western Blot identification,isothermal titration calorimetry?ITC?was used to detect binding of recombinant proteins VPAC1-EC1 and VPAC2-EC1 to VIP and PACPA27,respectively.The N-terminal extracellular domain?PAC1-EC1?recombinant protein of PAC1-R was prepared.ITC assay was used to complete the following detections:1)the binding of DOX/MINO with PAC1-EC1/VAPC?1/2?-EC1;2)the binding of SPAM1,a small molecule allosteric regulator targeting N-terminal extracellular domain of PAC1-R,to the N-terminal extracellular domain of PACAP three receptors;3)the competitive binding of PACAP?28-38?and TAT to PAC1-EC1.Results:The engineering strains pET30a-VPAC1-EC1-BL21?DE3?and pET30a-VPAC2-EC1-BL21?DE3?were successfully constructed.The proteins were expressed as inclusion body proteins.Through optimization of fermentation conditions,denaturation and renaturation,the yield of recombinant protein VPAC1-EC1 was 93±2mg soluble protein/L fermentation broth and VPAC2-EC1 was 78±5mg soluble protein/L fermentation broth.ITC measurements showed that the binding constant Ka of VIP and VPAC1-EC1 was 3.48E5±1.88E5M^-1,and Ka of PACPA27and VPAC2-EC1 was 5.88E6±3.06E6M^-1,indicating that VPAC1-EC1/VPAC2-EC1 had biological activity.The results of ITC assays were as followed:1)The binding constant Ka of VPAC1-EC1 and DOX was 2.58E5±1.49E5M^-1,the binding constant Ka of VPAC1-EC1 and MINO was 1.93E4±1.89E4M^-1;the reaction curve of VPAC2-EC1 and MINO could not be fitted;the binding constant Ka of VPAC2-EC1 and DOX was 2.86E6±7.99E5M^-1.The results showed that VPAC1-R N-terminal extracellular domain could bind to DOX/MINO,and VPAC2-R N-terminal extracellular domain bind to DOX with strong affinity.2)ITC tests verified that SPAM1 binds to PAC1-EC1,and the binding constant Ka is 2.59E6±1.90E6M^-1.SPAM1 does not bind to VPAC1-EC1,while SPAM1 binds to VPAC2-EC1with the Ka value of 3.09E5±2.87E5M^-1.3)ITC also detected the affinity of both PACAP?28-38?and TAT to PAC1-EC1,with Ka values of 5.03E5±1.59E5M^-1 for PACAP?28-38?and 6.35E5±8.39E4M^-1 for TAT.The subsequent competitive experiment verified that PACAP?28-38?and TAT recognized the same binding site on PAC1-EC1.Conclusion:Based on the successful preparation of recombinant N-terminal extracellular proteins of VPAC1-R/VPAC2-R,the binding of small molecules including DOX/MINO/SPAM1to VPAC1-EC1/VPAC2-EC1/PAC1-EC1 was detected by ITC for the first time.The competitive binding of PACAP?28-38?and transmembrane peptide TAT with N-terminal extracellular domain of PAC1-R targeting was detected by ITC for the first time.The above study lays experimental foundation for the in vitro screening and identification of agonists or regulators targeting N-terminal extracellular domain of three PACAP receptors.