The Underlying Roles And Mechanisms Of Porf-2 In The Transduction Of EphB1 Signals To Mediate Axon Growth

Background:Porf-2 was initially discovered as a growth-regulating factor in the central nervous system(CNS).It has been reported that Porf-2 was involved in the axon guidance of lower animals and dendritic spine formation and mutation of the mammalians.Due to the multiple alternative splicing variants,which have not been confirmed yet,the function of Porf-2 is rarely investigated.Our current study focus on deciphering the role of Porf-2 in axon growth and its underlying molecular mechanism,which will provide an avenue into the treatment axon regeneration-related diseases.Objective:1.To identify of the Porf-2 proteins in mice and its expression pattern.2.To investigate the role of Porf-2 in axon growth and its function domain.3.To screen and confirm the potential interacting proteins with Porf-2.4.To study the roles and the underlying mechanism of Porf-2 in different conditions of axon growth.Methods:1.Identification of Porf-2 protein:A combined Porf-2 plasmid followed by HA tag was constructed.The Porf-2 protein was then identified using specific anti-Porf-2 and anti-HA antibodies.The expression of Porf-2 was investigated by WB and immunofluorescence assay.2.The effect of Porf-2 on axon growth:The knockdown,overexpression and mutation of Porf-2 plasmids were used to detect the role of Porf-2 on axon growth and it potential molecular mechanism.3.Screening the Porf-2 interacting proteins:A combined Co-IP and mass spectrometry assay was used to detect the potential Porf-2-interacting proteins.Then the in vivo and in vitro Co-IP assay were used to confirm the interaction.4.The role of Porf-2 in the intrinsic axon extension and ephrin-B2-EphB1-mediated axon growth:The WT and EphB1 KO neurons were cultured from EphB1^+/+and EphB1^-/mice,of which the axon morphology was compared.The axon morphology change was assessed after ephrin-B2-Fc treatment in sh Ctrl-,sh Porf-2-and?GAP-Porf-2-transfected neurons.Results:1.The full-length Porf-2 protein about 130 kD,which is highly and specifically expressed in brain but almost no expression in other organs.2.Porf-2 can suppress axon growth,while?GAP-Porf-2 lost its inhibitory role.3.The binding ability between Porf-2 and EphB1 was increased in response to external ephrin-B2-Fc stimulation.Whereas,EphB1-?C failed to bind to and recruit Porf-2after ephrin-B2-Fc treatment.4.Overexpression of Porf-2 but not?GAP-Porf-2 inhibited the axon growth of both EphB1^+/+and EphB1^-/-neurons.5.Upon ephrin-B2-Fc treatment,both the axon growth were significantly reduced in cultured neurons.However,neurons transfected with sh Porf-2 or?GAP-Porf-2 failed to respond to ephrin-B2-Fc and exhibited a greater neurite length.Conclusions:1.Porf-2 protein is about 130 kD and highly expressed in brain.Porf-2 plays an inhibitory role in axon growth.2.The inhibitory function of Porf-2 on axon growth is exerted through Rac1/F-actin cytoskeletal signals via its GAP domain.3.Porf-2 can response to the activated EphB1 forward signals and is recruited by the intracellular part of EphB1.4.During initial axon growth,Porf-2 plays a restrictive role to prevent potential excessive growth in an EphB1-independent cell-autonomous manner.5.During axon-cell contact-mediated axon growth,Porf-2 serves as a downstream effector of EphB1 and transduces EphB1-dependent cytoskeletal dynamics to mediate axon growth and guidance.