Structural Study Of Deaf-related Protein VLGR1

VLGR1(The very large G protein coupled receptor 1)is currently the largest known G protein coupled receptor(GPCR).Its mRNA is 19 kb,which encodes 6299 aa.In cochlea,VLGR1 locates in the root of hair cells,and it's the core component of the ankle link complex.In mice,the mutation of Vlgrl results in abnormal stereociliary development and hearing loss,and the mutation of VLGR1 in human can lead to Usher syndrome and epilepsy.Early studies in our lab had shown that VLGR1 was cleavaged into two fragments after auto-cleavage at its G protein-coupled receptor proteolytic site(GPS site):? subunit and ? subunit.And the ? subunit(V?)inhibited adenylate cyclase activity by coupling G?i.Taken together,VLGR1 plays an extremely important role in the maintenance of ciliary morphology and auditory signal transduction during the development of the auditory system.Although significant progress has been made in the research of VLGR1,the molecular mechanism of intracellular signal transduction and its pathogenesis remain elusive,and its protein structure has not yet reported.Up to now,there are eight kinds of splicing isoforms of VLGR1,and VLGR1a,which is one of isoforms,as our research object.We have acquired Va via mutation at VLGR1a GPS site to prevent the hydrolysis of VLGR1a.Firstly,we fused cytochrome b562-RIL(BRIL)and T4 lysozyme(T4L)respectively in the intracellular loop 3(ICL3)of Va to explore the structure of Va.However,the expression detection of Va ICL3-BRIL and Va ICL3-T4L in Hek 293 cells was not ideal.Then we constructed the related fusion plasmids of Va-Gai and V?-Gai to insect cell system vector,which were expressed in sf9 cells,and got enough and homogenous protein sample by purification.Finally,we attempted to explore the complex structure of VLGR1and G protein via cryo-EM.Now,we have acquired the fusion protein of Va-Gai.And receptor particles with long strip,which are postulated to be calx-? of Va,can be observed preliminarily under electron microscope via negative staining.And we can see the different states of the receptor from the two-dimensional average results.While it is necessary to improve the expression and homogeneity of protein samples.In addition,we have studied the structure of V?-G?i2,in complex with G?/cherry-G?,and we can observe the existence of the complex under cryo-EM,then we can see the different morphology of the complex from the two-dimensional average results,while we also see monomer and dimers of the receptor.As the high requirement of cryo-EM to the concentration,purity and homogeneity of the samples,we need to optimize the conditions of expression,purification and the formation of the complex.In conclusion,our results are the foundation to exploring the three-dimensional structure of VLGR1,which will promote the research on its functional mechanism,and contribute to developing drugs for treating deafness.