Regulation Of Nociceptive Receptor TRPV2 By Protein-Serine/Threonine Kinase ARaf

TRPV2 channel(Transient Receptor Potential Vanilloid 2)is a non-selective cation channel which belongs to the transient receptor potential channel family.TRPV2 is widely present in both nociceptive sensory neurons and other non-neuronal tissues.TRPV2 channel serves as a polymodal receptor,which responses to mechanical,temperature,osmotic pressure,growth factors,hormones and other stimuli,involved in maintaining the normal function of cells.The dysfunction of TRPV2 channel may lead to a series of disorders.There is a growing interest in the research on TRPV2 channel functional regulation because it is crucial in physiology and pathology.However,far little is known about TRPV2 specific agonists and inhibitors.The lack of research in this area has seriously hindered the advances of TRPV2 functional study.For this reason,the interaction between the TRPV2 channel and the endogenous protein is the key to understand the regulatory mechanisms of TRPV2.In this study,we selected the N-terminal of the TRPV2 channel as the screening bait protein.Firstly,we established the mouse dorsal root ganglion cDNA library.Next,yeast two-hybrid method was used to screen the endogenous interaction proteins of dorsal root ganglion with TPRV2 channel.Finally,36 positive candidate genes were acquired through yeast two-hybrid screening,gene sequencing and bioinformatics analysis.It is known that reversible protein phosphorylation regulates many of the biological functions of eukaryotes,such as mediating protein-protein interaction,modulating enzyme activity,controlling subcellular localization,and affecting protein stability.It has been reported that the activity of ion channels can be regulated by protein reversible phosphorylation,so we selected ARaf(Serine/threonine-protein kinase ARaf)for further study.ARaf acts as a serine/threonine protein kinase,which plays a key role in cell function regulation by phosphorylation of downstream signaling molecules to transmit signals from membrane receptors into cell,such as cell proliferation,differentiation and transformation.A series of experiments were carried out for whether ARaf can regulates TRPV2 channel function by phosphorylation.The results are summarized below:l)The interaction of ARaf and N-terminal or full length of TRPV2 channel was confirmed by yeast rotation and Co-Immunoprecipitation(Co-IP).Furthermore,Fluorescence confocal showed a co-localization of Co-expression of TRPV2 and ARaf in Hek293T cells.2)Immunoblotting showed that the presence of ARaf significantly increased TRPV2 serine and threonine phosphorylation levels.3)In addition,the sensitivity of TRPV2 channel to agonist 2-APB was decreased and the concentration-dependent curve shifted to the right when TRPV2 channel and ARaf were co-expressed in Hek293T cells on the whole-cell patch-clamp test.4)Site-directed mutagenesis screening showed that TRPV2 S65A,S466A,T522A,S526A had no significant change in the sensitivity of TRPV2 to 2-APB when ARaf exists,which indicated that S65,S466,T522,S526 residues may be TRPV2 effector sites of ARaf phosphorylation.The above results indicate that ARaf down-regulates TRPV2 channel activity by phosphorylation and we find four potential phosphorylation amino acid residues located on the inner side of the membrane of TRPV2.These findings enrich the the functional adjustment means of TRPV2 channel and provide a guidance for other similar research.Further more,the other candidate proteins obtained by the yeast two-hybrid screening lay a good foundation for the follow-up study.