Expression And Purification Of Angiotensin Ⅱ Type 1 Receptor Heterodimers

G Protein Coupled Receptors(GPCRs)are important drug targets that can exist and function as dimer or oligomer under physiological conditions.Among them,heterodimers have unique biochemical characteristics and pharmacological effects,including receptor trafficking,ligand binding,and signaling,which are different from GPCR monomers.They also involve in human disease.However,the drug development targeting GPCR heterodimers was slow.Therefore,we need structural information of GPCR heterodimers to elucidate the relationship between receptor heterodimerization and functional alterations as well as drug design.The Angiotensin II Type-1 Receptor(AT1R)is an important member of the class A GPCRs,mediating most of the physiological effects of Ang II in human.It helps with the cardiovascular function regulation and human fluid homeostasis.It’s an important drug target for heart failure and hypertension.AT1 R can form heterodimers with a variety of GPCRs and perform unique functions.When AT1 R dimerizes with Angiotensin II Type-2 Receptor to form AT1R-AT2 R heterodimer,they inhibit the AT1R-mediated Gq signaling and antagonizes the function of AT1 R.AT1R-AT2 R heterodimer is a potential drug target for diseases including gestational hypertension and Parkinson.Similarly,AT1R-Mas heterodimerization also reduces AT1R-mediated phosphatidylinositol production and intracellular calcium mobilization.In contrast,AT1R-B2 R heterodimerization would result in Ang II hypersensitivity and enhance AT1R-mediated signal,contributing to human pregnancy hypertension.AT1 R signal sensitization of patients with preeclampsia was attributed to AT1R–B2R dimerization.Although evidence have shown the existence of AT1 R heterodimers and their association with human disease,there’s no drugs targeting AT1 R heterodimers available due to the lack of AT1 R heterodimer structures and the dimerization mechanism.In addition,there isn’t any structural biology study of AT1 R heterodimers and class A GPCR heterodimers for reference.The bottleneck of class A GPCR heterodimers’ structural biology study is the difficulty of obtaining stable protein samples through purification.Therefore,in order to determine the structures of AT1R-AT2 R heterodimer,AT1R-Mas heterodimer and AT1RB2 R heterodimer,we expressed the proteins of those heterodimer in insect cells Sf9 using baculovirus expression system.Because of the flexibility and the instability of the heterodimers,we try using the Nano Bi T method,chemically induced dimerization(CID)method,lipid Nanodiscs method and other strategies to promote and stabilize heterodimerization.We also tried to improve the expression and stability of the dimer by means of fusion protein screening,receptor truncation,and amino acid mutation.Our results laid a foundation for the subsequent structural biology studies of AT1R-AT2 R,AT1R-Mas and AT1R-B2 R heterodimers and the structure-based drug development.For AT1R-AT2 R heterodimer,we chose FKBP-FRB-Rapamycin system of CID strategy and screen the constructs using terminal truncations method.We also screened the detergent,ligand,and rapamycin dosage in the protein purification process.The protein samples had high purity and good stability.The protein particles with good homogeneity and expected size were observed in the negative staining TEM images.Now,the efforts of purification process optimization are being made to maintain protein’s good homogeneity when preparing cryo-EM samples.For AT1R-B2 R heterodimer,we attempted to use nanobodies and downstream signaling proteins to increase its stability.After screening Nano Bi T method and CID method,we decided to use the latter one and attached the FKBP and FRB proteins to the two receptors respectively.We screened the insertion position of the purification tag,the terminal length of the two receptors,the amino acid mutation of AT1 R,and finally obtained appropriate samples with highest purity and better homogeneity.The screening of purification conditions is currently being carried out to obtain stable protein sample that can still be separated by size-exclusion chromatography(SEC)when scaling up the purification system.For AT1R-Mas heterodimer,the wide receptor was engineered and deglycosylated to obtain samples with suitable properties,but the results of protein purification amplification are still unknown.In summary,a variety of stabilizing methods were used to obtain the AT1R-AT2 R,AT1R-B2 R and AT1R-Mas heterodimers.Finally,through protein expression and purification in vitro,the above three heterodimers protein samples with high purity and good homogeneity were successfully obtained,laying an important foundation for the structural determination of AT1 R heterodimers by cryo-EM.The structural information will facilitate the functional study of AT1 R heterodimer and promote the development of drugs targeting AT1R heterodimer.