Mechanisms Underlying The Differential Ret And Trkb Surface Levels And Activity Enhanced Ret Surface Insertion

BackgroundThe RET tyrosine kinase is required for the development of the kidneys, testes, enteric, and nervous systems. In nervous system, RET expression and functions are well investigated in peripheral and sensory neurons. For instance, RET positive neurons comprise about a half of total adult DRG neurons, which are called non-peptidergic nociceptors, and RET is proposed to be critical for the development and maintenance of non-peptidergic nociceptors. Interestingly TrkB is also expressed in adult non-peptidergic DRG neurons and is essential for postnatal survival of non-peptidergic nociceptive neurons. The activation of Ret is stimulated by the glial cell line derived neurotrophic factor (GDNF) family ligands (GFLs). GFLs interact directly with Ret co-receptors known as GDNF family receptor alphal-4(GFRal-4), which then form active receptor complexes with Ret. GFL-mediated Ret activation stimulates multiple intracellular signaling pathways including RAS/MAPK and PI3K/Akt that promote cell survival, cell migration, and neurite outgrowth.Proper cell surface localization of RET receptor is crucial for its normal functions, however little is known about the modulation of RET surface expression. Increasing evidences indicate that complex arrays of short signals and recognition amino acid sequences ensure accurate trafficking of transmembrane receptors into cell membrane. In this process, protein kinases are usually involved. For example, it has been reported that PKC could facilitate NMDA receptor surface delivery. Neuronal activity could also enhance receptor surface insertion through activation of protein kinases. In nerve system, the activity dependent surface insertion of AMPA receptors is a well-researched model. However, it is still unknown whether such mechanisms are involved in the regulation of RET surface expression.Objective1. Compare the cell surface expression level of RET and TrkB receptors and find out is there a key amino acid motif charging for their surface expression.2. To find out whether protein kinase could modulate RET surface expression.3. Investigate whether the neuronal activity enhance RET surface insertion.4. Analyze the effect of modulating RET surface expression on RET mediates signaling and cell function.Methods and results1. Comparing of RET and TrkB cell surface level in cultured DRG neuronsWe confirmed RET and TrkB co-expression in P7newborn rat DRG neurons by immunohistochemical staining. Then we determined there surface expression levels in cultured DRG neurons by surface biotinylation methods. The results shown RET surface level was about1.5fold of TrkB.2. Cytoplasmic domains determine the differential RET and TrkB surface expression levelsChimeric receptors with switched extracellular domain of RET and TrkB were constructed (RETTrkBIC and TrkBRETIc) and transfected into PC12cells. By surface biotinylation assay, we found in condition of the same extracellular domain (RET or TrkB extracellular domain), RET and TrkB still exhibited differential cell surface levels, which suggested that the intracellular domains of RET and TrkB were responsible for their differential surface expression.3. Establishment of immunofluorescence staining methods for quantifying receptor surface expression levelsWe adopted a convenient and visible ratiometric fluorescence assay methods for relatively comparing cell surface receptor levels. In this assay, receptors are Flag-tagged at the N-terminal of extracellular domain and fused with green fluorescence protein (GFP) in the cytoplasmic c-terminal. Thus, cell-surface receptor levels can be quantified by fluorescence intensity of Flag staining normalized to GFP intensity per cell under non-permeabilized condition. When transfected into PC12cells, ratiometric fluorescence assay revealed that the surface receptor level of RET-GFP was-1.5fold compared with that of TrkB-GFP, which was consistent with the difference between RET and TrkB cell surface level under endogenous condition in DRG neurons.4. The Boxl motif is responsible for the differential RET and TrkB surface levelUsing sequence substitution strategy, we identified a key motif (Box1) which is necessary and sufficient for the differential RET and TrkB surface expression levels. Substituted RET Box1to TrkB significantly increased its surface level while Substituted RET Boxl motif by TrkB respective motif decreased the surface level of RET.5. PKC activity enhances RET surface expressionSequence/residue and protein kinases interaction plays an important role in the modulation of receptor surface expression. Inspection of RET Boxl sequence revealed a threonine residue (Thr675) confirming the PKC phosphorylation consensus sequence. Thus we test the effect of PKC activity on RET surface expression by pharmacological methods. PKC inhibitor CHE treatment down-regulated surface level of RET while PKC activator TPA increased it. RET-Thr to Ala mutant blocked the effect of TPA on RET surface level. These result suggested that PKC activity could enhance RET surface expression through phosphorylation on RET Thr67residue.6. Depolarization enhances RET cell surface expression through PKCNeuronal activity could induce the activation of intracellular protein kinases and modulate cell response to particular stimuli through regulation of cell surface receptor level. Increasing neuronal activity by50mM K+depolarization evaluated surface RET amount which could be blocked by PKC inhibitor CHE and Thr to Ala mutant.7. RET Thr675residue is phosphorylated by PKC and depolarizationTo confirm that Thr675in site in RET Box1motif is directly phosphorylated upon PKC activation or depolarization, we generated a new phospho-specific antibody against the RET Thr675site (rabbit anti-pT675RET). TPA and depolarization significantly increased Thr675phosphorylation. In contrast, CHE decreased Thr675phosphorylation and abolished the effect of depolarization. Thus, Thr675residue in RET specially served as a PKC phosphorylation site to modulate RET surface level.8. Thr675site in RET regulates ligand-induced signaling and cell differentiationModulation of cell surface receptor level is a well-established mechanism for controlling cell response to particular stimuli. Since we have found Thr675site was critical for modulating RET surface distribution, next we want to know whether the Thr675site might modulate GDNF-induced signaling pathway and cell response. In transfected PC12cells,, the phosphorylation levels of RETT675A and subsequent activation of Akt and Erk were significantly reduced compared with those of RET. GDNF induced cell differentiation percentage through RETT675A also reduced in comparing with RET. Therefore, Thr675site in RET Box1motif could modulate GDNF-induced RET signaling and cell differentiation, which might be due to its regulation of RET cell surface level.Conclusion1. RET and TrkB receptors exhibited differential surface expression levels and identified a key motif (Box1) in RET juxtamembrane region responsible for this difference.2. PKC activity and neuronal depolarization could enhance RET cell surface level through phosphorylation on the Thr675site in RET Box1motif.3. RET Thr675residue could regulate GDNF-induced signaling and cell differentiation.