Identification Of The Interaction Between Nerve Growth Factor Receptor TrkA And Signaling Molecule Mint2

Nerve Growth factor (NGF) is a member of the superfamily of neurotrophic factors that control the development and survival of certain neuronal populations both in the peripheral and in the central nervous system. TrkA, the high affinity NGF receptor, the molecular masses 140KD,contains an intracellular domain with tyrosine kinase activity. NGF binding to TrkA receptors results in receptor dimerization and kinase activation.Using the yeast two-hybrid system, Mint2 was found to bind to the cytoplasmic domain of TrkA. Mints are adaptor protein family with three members. Mint1 and Mint2 are expressed only in neural tissues, whereas Mint3 is ubiquitously expressed. All Mints share a conserved central PTB domain and two C-terminal PDZ domains. Although Mints was originally described and named for its ability to bind Munc18, a neuronal protein acting at the synapse, it also binds a number of other proteins, most notably the p-Alzheimer's precursor protein (P-APP), neurexins, and a number of transmembrane receptors. Recent data suggest that over-expression of the Mint2 prolongs the half-life of APP and enhances APP expression level. A is derived from the amyloid precursor protein. AP40 and Ap42 are released by P- and secretase activities that cleave APP at the amino and carboxyltermini of Ap respectively. Increased production and deposition of the 40-42-amino acid amyloid peptide (AP) is believed to play a key role in the pathogenesis of Alzheimer's disease.Using yeast two-hybridization and co-immunoprecipitation and GST-Pull down assay, in present study, we have identified interaction of signaling moleculer Mint2 with NGF receotor TrkA in vivo and in vitro. The main results of our research are as follows:The intracellular part of TrkA was fused to LexA and used as a bait plasmids( pGilda-TrkAIC ). The cDNAs encoding the amino-terminal region of Mint2, PTB domain, PDZ1 domain, PDZ2 domain and the entire sequence of Mint2 were subcloned into pB42AD. These plasmids were used for the TrkA10 binding assay inco-transfections with pGilda-TrkA into yeast cells HLY819 respectively. Theinteraction was confirmed by using p-galactosidase activity assay in yeast. Ourresults showed that the PTB domain was capable of strong interaction with TrkA as well as full length of Mint2. Although the PDZ1 domain and PDZ2 domain could bind to the TrkAIC, the interaction was very weak. The ammo-terminal region of Mint2 was incapable of interaction with TrkAIC in yeast two-hybrid system.The cDNAs encoding the amino-terminal region, PTB domain, PDZ1 domain and PDZ2 domain of Mint2 were amplified by PCR and inserted into the expression vector pGEX-4T-3 and transformed into E.coli BL21(DE3). All constructs were confirmed by restriction analysis and sequencing. The SDS-PAGE analysis revealed that the proteins were highly expressed after IPTG induction. The solubilized recombinant GST fusion proteins were purified by affinity chromatography. To identify which domain of Mint2 binding to TrkA, we carried out a GST-pull down assay by using the purified recombinant GST fusion proteins. Our finding indicatedthat the PTB domain was capable of interaction with TrkA in vitro. At the same time, the data suggested that the PDZ1 domain and PDZ2 domain could also bind to TrkAIC in vitro, but the binding affinity between them was weak compared with the PTB domain. In addition, the amino-terminal region of Mint2 did not bind to TrkAIC in vitro.To determine an ability of Mint2 to associate with TrkA in vivo, we performed immunoprecipitation experiment with homogenates from rat forebrain, cerebellum and spinal cord. Protein lysates were immunoprecipitated with antibody against TrkA and the immunocomplexes were probed with anti-Mint2. The Mint2 protein was detected in the immunocomplexes from forebrain, spinal cord homogenates but not in cerebellum homogenates, as few TrkA was probed in protein lysates from cerebellum homogenate. To confirm further the association, we immunoprecipatated the protein lysates with anti-Mint2 and probed the immunoblot w...