Study On The Mechanism Of Conformational Changes And Functional Relationship During The Activation Of Integrin ?_?b?₃

The research on the inside-out activation mechanism of integrin mainly focuses on the intracellular region of ?-integrin,which is relatively conservative and is also the binding site of the activator talin and kindlin.The proximal region of the intracellular region of ?-integrin and?-integrin share a conserved GFFKR motif and form a specific interface to keep integrin in a resting state.There are differences in the length and sequence of the distal membrane end region of?-integrin after the GFFKR motif,and its roles in integrin activation have not been determined.In this experiment,the ??b and ?3 subunits were truncated and mutated,and the distal cytoplasmic membrane region of the integrin was studied through experiments such as soluble ligand binding,LIBS epitope exposure,disulfide bond cross-linking,and WB.This study confirmed that the distal cytoplasmic membrane region of ?-integrin helps maintain the resting state of integrin and inside-out activation of integrin.The complete deletion of the distal region of ?-integrin diminished the binding and conformational changes of integrin ligand mediated by talin and kindlin.Inside-out activation of integrin requires that the distal membrane end region of ?-integrin has the proper length and the proper amino acids.Experiments have confirmed the important role of ?-integrin cytoplasmic distal region in integrin activation.It also provides new insights into how talin and kindlin induce conformational changes of integrins.The ?1/?1'-helix in the extracellular region of integrin is bent at the ?1/?1' junction without ligand binding.After the integrin binds to the ligand,the al/al'-helix changes from the curved conformation to the straightened conformation in the ?I domain where the ligand is bound.In order to determine whether high-affinity ligand binding and bidirectional conformational conduction of intact integrins on the cell surface require conformational changes in the?1/?1'-helix,this experiment,mutations were introduced at the junction of ?1/?1' in the extracellular region,and they were studied by experiments such as soluble ligand binding,LIBS epitope exposure,cell adhesion and inhibition,and cell extension.Experiments have proved that there is a conserved glycine at the ?1/?1' junction,which plays an important role in the curved conformation of the ?1/?1'-helix in the resting state.Mutations that promote ?1/?1'-helix extension maked integrin structurally active.However,in external or internal stimulation,the mutation that blocks the ?1/?1'-helix from bending does not bind to the soluble ligand.These mutations also prevented the ligand-induced extracellular extension of integrin,but have no effect on the talin-induced extension.In addition,the ability of these mutant integrins to mediate cell proliferation,F-actin stress fiber and focal adhesion formation,and focal adhesion kinase activation were reduced.The experiment also confirmed the structural role of the ?1/?1'connection,which makes the ?1/?1'-helix bent in a resting state,and transmits a bidirectional conformation signal through the helix stretch when the integrin is activated.