Trafficking of TrkA-Green Fluorescent Protein Chimerae during Nerve Growth Factor-induced Differentiation*

Jérôme JullienDagger §, Vincent GuiliDagger §, Edmund A. Derrington, Jean-Luc Darlix, Louis F. Reichardt||**, and Brian B. RudkinDagger DaggerDagger

From the Dagger  Differentiation and Cell Cycle Group, Laboratoire de Biologie Moleculaire et Cellulaire, UMR 5665 CNRS, Ecole Normale Supérieure de Lyon and  LaboRetro, INSERM U412, Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, 69364 Lyon Cedex 07, France and || Howard Hughes Medical Institute, University of California, San Francisco, California 94143

Received for publication, March 12, 2002, and in revised form, November 14, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A chimera of the nerve growth factor (NGF) receptor, TrkA, and green fluorescent protein (GFP) was engineered by expressing GFP in phase with the carboxyl terminus of TrkA. TrkA-GFP becomes phosphorylated on tyrosine residues in response to NGF and is capable of initiating signaling cascades leading to prolonged MAPK activation and differentiation in PC12 nnr5 cells. TrkA constructs, progressively truncated in the carboxyl-terminal domain, were prepared as GFP chimerae in order to identify which part of the receptor intracellular domain is involved in its trafficking. Immunofluorescence observations show that TrkA-GFP is found mainly in cell surface membrane ruffles and in endosomes. Biochemical analysis indicated that the cytoplasmic domain of TrkA is not necessary for correct maturation and cell surface translocation of the receptor. An antibody against the extracellular domain of TrkA (RTA) was used as ligand to stimulate internalization and phosphorylation of TrkA. Co-localization studies with anti-phosphorylated TrkA antibodies support a role for such complexes in the propagation of signaling from the cell surface, resulting in the activation of TrkA in areas of the endosome devoid of receptor-ligand complexes. Confocal time-lapse analysis reveals that the TrkA-GFP chimera shows highly dynamic trafficking between the cell surface and internal locations. TrkA-positive vesicles were estimated to move 0.46 ± 0.09 µm/s anterograde and 0.48 ± 0.07 µm/s retrograde. This approach and the fidelity of the biochemical properties of the TrkA-GFP demonstrate that real-time visualization of trafficking of tyrosine kinase receptors in the presence or absence of the ligand is feasible.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Growth factors of the neurotrophin family are involved in the survival and differentiation of certain neurons in the peripheral nervous system (1). Signaling by these proteins is believed to be mediated by binding to the neurotrophin receptor p75NTR and/or to one member of the Trk family. p75NTR binds to each neurotrophin with similar affinity. By contrast, Trk receptors exhibit differential affinity toward neurotrophins: TrkA binds preferentially to NGF,1 TrkB to BDNF (brain-derived neurotrophic factor) and NT4-5, and TrkC to NT-3 (2, 3).

Trk family members are tyrosine kinase receptors. Signaling by the NGF-TrkA complex is the best characterized. Upon NGF binding to the receptor, the tyrosine kinase domain of TrkA is activated, resulting in the autophosphorylation of two major tyrosines in the intracellular domain of the receptor (4). These phosphotyrosines are docking sites for several adaptor proteins that are involved in signal transduction pathways leading to the activation of MAPK, phosphatidylinositol 3-kinase, and phospholipase C-gamma (5). By contrast, p75NTR does not present any known catalytic activity in its cytoplasmic domain. Recent studies have reported the identification of several proteins that bind to the cytoplasmic domain of p75NTR (6-9). Depending on the interactor considered, binding of a given effector to p75NTR has been shown to be either enhanced or decreased by NGF binding. The precise roles of these proteins in the mediation of NGF signaling remain to be elucidated.

In the case of neurons, additional specific spatial constraints for neurotrophin receptor signaling must be considered. The neurotrophin hypothesis postulates that the source of NGF for neurons is restricted to the target field of innervation (10). Most often, cell bodies of neurons are located far from their target cells. Thus, neurotrophin is available only at the growth cone of the neuron. NGF-induced survival requires transmission of a signal from the axon tip to the cell body where transcriptional modifications are induced. Numerous studies indicate that the transmission of this NGF survival effect in neurons of the peripheral nervous system is supported by retrograde transport of NGF-TrkA complexes from the neurite tip to the cell body (11-14). This points to the importance of intracellular trafficking of neurotrophin receptors in the implementation of a response to their ligands. Indeed, NGF neuronal responsiveness is highly dependent upon NGF receptor targeting to where the growth factor is provided, namely the axon tip. Once complexed to its ligand, activated receptor is transported back to the cell body. Some of the intracellular trafficking steps underlying this process have been elucidated. Retrograde transport is initiated by internalization via the coated pit pathway (11, 15-17). After this step, TrkA is believed to undergo a microtubule-supported vesicular retrograde transport to the cell body (10, 12).

Internalization by coated pits appears to require trafficking motifs in the intracellular domain of internalized proteins. Two major trafficking motifs have been identified (18, 19). These motifs are short stretches of amino acids corresponding to either the dileucine type (two adjacent leucines or a leucine and an isoleucine) or to the tyrosine-based type (YXXphi or NPXY type, where X is any amino acid, and phi  is a hydrophobic amino acid). In addition to their involvement in the internalization processes, these trafficking motifs appear to mediate the recruitment of a receptor along numerous trafficking pathways ranging from targeting to specific cell surface domains to accumulation in defined intracellular sites (19-21). Signal-mediated protein targeting is believed to be supported by recognition of these motifs by members of the adaptor complex family.

The aim of the present study was to gain insight into TrkA trafficking steps prior to and during exposure to NGF and the resulting differentiation process. The intracellular domain of TrkA contains numerous potential trafficking motifs of the tyrosine-based and dileucine types. Involvement of these motifs in TrkA trafficking has been investigated using receptor chimerae designed with TrkA progressively truncated from the carboxyl terminus in phase with GFP. Data are presented indicating that fusion of GFP to full-length TrkA does not modify the expected functioning of the receptor. Cell surface translocation together with NGF-induced internalization of receptor chimera have also been analyzed to ascertain which parts of the TrkA cytoplasmic domain are involved in trafficking of the receptor. Confocal time-lapse analysis of TrkA-GFP trafficking during NGF-induced differentiation was undertaken to visualize and evaluate the kinetics of the actual trafficking of the receptor. A description of TrkA localization at different phases of differentiation is presented together with the major fluxes occurring between cellular compartments during this process.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents-- Sulfo-NHS-biotin and streptavidin-agarose were purchased from Pierce. Anti-TrkA extracellular domain (RTA) was prepared as described previously (22). Anti-transferrin receptor (HTR68-4) was kindly provided by I. Trowbridge (The Salk Institute). Anti-phospho-490 Trk (E6) and anti-phosphotyrosine (PY99) were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-EGFP (JL-8) was from Clontech. Anti-active MAPK (anti-ERK 1/2 pAb, catalog No. V1141) was from Promega. R-phycoerythrin-conjugated Affinipure F(ab')2 fragment donkey anti-rabbit IgG, rhodamine-conjugated anti-mouse IgG, cyanine-5-conjugated anti-mouse antibody, and phalloidin-rhodamine were from Jackson Immunoresearch laboratories, Inc. NGF from mouse submaxillary glands was from Quality Controlled Biologicals. Protein A-Sepharose 4 fast flow was from Amersham Biosciences. K252-a was from Calbiochem, and Polybrene was from Sigma.

Constructs-- DC971 retroviral vector encoding rat neuronal TrkA has been described (22). An EGFP insert was prepared by PCR reaction using GFP-ol5' and GFP-ol3' primer, allowing the introduction of ClaI site in 3' of EGFP stop codon and StuI and NsiI site in 5' of EGFP start codon (GFP-ol5', 5'-ATAGGCCTTG ATGCATATGGTGAGCA AGGGCGAGG-3'; GFP-ol3', 5'-CCATCGATTT ATCTAGATCC GGTGGATC-3'). The Delta 1 chimera is the result of EGFP ligation between the ClaI and StuI sites of DC971. This ligation create an in-frame fusion between the EGFP amino terminus and a truncated TrkA carboxyl terminus missing 4 amino acids of TrkA carboxyl terminus. Full-length TrkA-EGFP fusion (Delta 0) was reconstituted by ligating prehybridized End1 and End2 complementary oligonucleotides between the NsiI and StuI sites of Delta 1 (End1, 5'-CCTTGGCACA GGCGCCACCG AGTTACCTGG ACGTTCTGGG CATGCA-3'; End2, 5'-TGCCCAGAAC GTCCAGGTAA CTCGGTGGCG CCTGTGCCAA GG-3'). Other truncated constructs were produced using the kit Erase-a-Base (Promega). ExoIII digestion was performed on an NsiI/StuI-digested Delta 0 plasmid, and all subsequent steps were performed following user guide instructions. Kinase-dead Delta 0 (Delta 0-KD) corresponds to a Delta 0 construct carrying a point mutation, K547N, which inactivates the kinase activity of TrkA. This construct was obtained by mutating pLNCX-Delta 0 using the Transformer mutagenesis kit (Clontech) with the following primers: Select-ol, 5'-CATCATTGGAA AACGCGTTTC GGGGCGAAAA CTC-3'; K547N-ol, 5'-GCTGGTGGCT GTCAACGCAC TGAAGGAGAC ATC-3'. All of the constructs were verified by sequencing (ABI Prism kit, Applied Biosystems, Foster City, CA). pcDNA3-Delta ECTO was generously provided by Dr. A. Pandiella (Instituto de Microbiologia Bioquimica, Salamanca, Spain). Delta ECTO insert was extracted from pcDNA3 vector and subcloned into the pIRES-2 EGFP vector from Clontech.

Cell Surface Biotinylation and Immunoprecipitation-- Intact cells were incubated for 45 min in ice-cold PBS containing 0.5 mg/ml sulfo-NHS-biotin. Cells were then washed four times in PBS supplemented with 2 mM lysine to remove unbound reactive biotin. After cell surface biotinylation, proteins were extracted using ice-cold lysis buffer (20 mM Tris-HCl, pH 8, 137 mM NaCl, 2 mM EDTA, 10% glycerol, 1% Nonidet P-40, 20 µM leupeptin, 1 mM sodium vanadate, 1 mM Pefabloc, 0.15 units/ml aprotinin, 1 mM beta -glycero-phosphate, 6 mM sodium-fluoride). The extract was clarified by centrifugation at 12,000 × g for 10 min. Cleared lysate was then subjected to precipitation with streptavidin-agarose beads to obtain the cell surface fraction. In TrkA-GFP immunoprecipitation experiments, cleared lysates were precipitated using the JL-8 antibody directed against EGFP. Immunocomplexes were collected using protein A-Sepharose beads and eluted by boiling for 10 min in sample buffer. Proteins were then subjected to SDS-PAGE and Western blot analysis.

Flow Cytometric Analysis-- Cells were collected at 37 °C in PBS supplemented with 0.1 mM CaCl2, 1 mM MgCl2, and 5 mM EDTA. After centrifugation cells were resuspended in culture medium and subjected to 50 ng/ml NGF treatment at 37 °C on a rotating wheel. Cells were then washed twice in ice-cold PBS and used as "live intact cells" for immunolabeling. All of the subsequent labeling steps were performed on ice. Cells were washed twice in blocking buffer (PBS with 0.5% bovine serum albumin and 0.02% sodium azide) and incubated for 30 min in the same buffer containing an antibody directed against RTA. After two washes in blocking buffer, cells were exposed to phycoerythrin-labeled secondary antibody for 30 min and washed three more times. Cells were then analyzed using a FACScan flow cytometer (BD Biosciences) equipped with an argon ion laser tuned to 488 nm. Emission fluorescence was measured with a 585-42-nm dichroic filter (phycoerythrin fluorescence) or a 530-30 bandpass filter (EGFP fluorescence). Data acquisition and analysis were performed with CellQuestTM software (BD Biosciences). Internalization efficiency was calculated from the amplitude of the shift in the geometric mean of the phycoerythrin signal between treated and untreated GFP-positive cells.

Cell Culture-- PC12 cells, PC12 nnr5 cells (obtained from Dr. P. Barker; Montreal Neurological Institute), and PC12 6-24 cells over-expressing human neuronal TrkA (provided by Dr. D. Martin-Zanca, Instituto de Microbiologia Bioquemica, Universida de Salamanca, Spain) were grown as described previously (23).

Transfection and Infection-- Transfections were performed using the calcium phosphate precipitation (23). Helper cell lines (TE-FLY (53), generously provided by Dr. F.-L. Cosset, Lyon, France) were transfected with retroviral vector encoding TrkA-GFP constructs and selected with G418 for 3 weeks. Stably transfected helper cells, in the exponential phase of growth, were incubated for 12-16 h in complete PC12 cell medium. Vectors were prepared by filtering the medium on a 45-µm filter. PC12 nnr5 or HeLa cells were pretreated for1 h with 12 µg/ml Polybrene and then incubated for 8 h in the presence of filtered vector. After three rounds of infection, cells were selected for neomycin resistance.

Confocal Microscopy-- PC12 cells were transfected with TrkA-GFP vector. At 24 h post-transfection, cells were spread on collagen poly-L-lysine-coated coverslips (24). At 48 h post-transfection, cells were fixed for 10 min in PBS, 3.7% formaldehyde and permeabilized for 1 min in PBS, 0.5% Triton X-100. After being washed with PBS, cells were blocked with PBS, 0.5% bovine serum albumin for 30 min and incubated with primary antibody for 30 min. Cells were washed three times in PBS and incubated for an additional 30 min with secondary antibody. After washing, cells were mounted in Moviol. Scanning fluorescence images were acquired using the MRC1000 confocal laser unit (Bio-Rad) coupled to a Zeiss Axioplan microscope equipped with a Zeiss X40, C-APO, 1.3 NA oil immersion objective.

Confocal Time-lapse Microscopy-- Confocal time-lapse fluorescence images were acquired using the MRC1000 confocal laser unit (Bio-Rad) coupled to a Zeiss Axiovert microscope equipped with a Zeiss X40, C-APO, 1.3 NA oil-immersion objective. Cells were maintained at 37 °C, and analyses were performed in a CO2-independent medium (Invitrogen) to avoid medium acidification in a CO2-free atmosphere. GFP bleaching experiments were performed using the photobleaching module of LSM 510 software (Zeiss) with a 488-nm argon laser set to 100% power. In vesicle tracking experiments, pinholes of the confocal unit were set so that the optical slice obtained encompassed the entire thickness of the structure studied. Unless specified, pinhole settings were ~1 airy/unit.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Fusion of GFP to TrkA Carboxyl Terminus and Truncated Mutants-- A TrkA-GFP chimera, named Delta 0, was engineered by the addition of EGFP to the carboxyl terminus of rat neuronal TrkA (Fig. 1A). Deletion mutants of TrkA-GFP with sequential truncation of the cytoplasmic domain of the receptor were constructed to test the potential involvement of the dileucine and tyrosine-based motifs in trafficking of TrkA (Fig. 1A). Fig. 1B indicates the terminal amino acid of each TrkA deletion mutant, the potential trafficking motif that was removed with respect to the previous construct, the theoretical kinase activity based on published information on structure activity, and finally, the tyrosine involved in the known signaling pathways still present in the construct. In the following presentation, TrkA-GFP will be used to refer to all of the truncated chimeric receptors, whereas Delta n will refer to the fusion of GFP with a specific truncated TrkA mutant missing n potential trafficking motifs (Fig. 1A).


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Fig. 1.   TrkA-EGFP chimerae and associated mutant. A, schematic representation of TrkA-GFP constructs. Extra, extracellular domain; TM, transmembrane domain; Intra, intracellular domain. L and Y are, respectively, dileucine and tyrosine-based trafficking motifs. The number attributed to each deletion refers to the number of potential trafficking motifs deleted. B, characteristics of truncated TrkA-GFP mutants. All mutants have the signal peptide at the amino terminus. For each mutant, the first amino acid of the remaining TrkA carboxyl terminus is specified. Potential trafficking motifs eliminated by each deletion are also indicated. The tyrosines Tyr794 and Tyr 499 of rat TrkA are believed to support the NGF-induced recruitment of signaling molecules to the receptor. The presence of these tyrosines in the deletion mutants is also indicated. The kinase activity of the receptor is deduced from the presence of an intact kinase domain in which the phosphorylation loop includes Tyr764, Tyr 765, and Tyr 777 or from published data (Delta ECTO (55)).

Trafficking Properties of TrkA-GFP-- The expression and cellular localization of the various constructs were initially characterized in the absence of NGF. Particular attention was given to the analysis of cell surface targeting of TrkA because this event is crucial in determining cellular responsiveness to NGF.

Intracellular Localization-- Confocal microscopy observations indicate that Delta 0 is targeted essentially to two cellular locations at steady state in the absence of NGF. Delta 0 accumulates mainly at an internal perinuclear location and is also found at the cellular periphery in the form of discrete bright punctate structures (arrows in Fig. 2, D, G, and J, respectively). Double labeling experiments with the endoplasmic reticulum marker calreticulin indicate that the relatively faint diffuse intracellular TrkA labeling corresponds to receptors located in the endoplasmic reticulum (ER) (arrows in Fig. 2, A-C). The intracellular perinuclear compact labeling of Delta 0 may be attributed, at least in part, to an endosomal location, as shown by co-localization with the endosomal marker Tnf-R (Fig. 2, D-F). Cell surface TrkA has been detected by immunolabeling unpermeabilized cells with an antibody directed against the TrkA extracellular domain. The cell surface TrkA labeling co-localizes with peripheral Delta 0-positive dots (Fig. 2, G-I). Thus, it appears that the Delta 0 peripheral labeling reflects the localization of cell surface TrkA. These discrete Delta 0 cell surface structures also appear to co-localize with peripheral actin, a well known marker of membrane ruffles (Fig. 2, J-L). The same kind of confocal microscopy analysis was undertaken with TrkA-GFP deletion constructs. Characteristic patterns of distribution are presented in Fig. 3. Delta 0 and kinase-dead Delta 0 (Delta 0-KD) exhibit strong endosomal expression, Delta 2 more diffuse cellular endoplasmic reticulum distribution, and Delta 8 very weak endosomal location with strong membrane expression. The results of these experiments are summarized in Table I. This study shows that Delta 0, Delta 1, and Delta ECT appear to accumulate in the perinuclear endosomal compartment at much higher levels than other constructs. It appears that all of the truncated TrkA constructs, with the exception of Delta TM and Delta ECT, are targeted to the cell surface membrane ruffles of PC12 cells. A biochemical study was undertaken to analyze the maturation and cell surface targeting of TrkA constructs at a molecular level.


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Fig. 2.   Cellular localization of TrkA-GFP (Delta 0) chimera. Intracellular localization of TrkA was investigated in transiently transfected PC12 cells by confocal microscopy. Double labeling experiments were undertaken to analyze putative co-localization between TrkA-GFP (Delta 0, green) and several intracellular compartments (in red). The endoplasmic reticulum and endosomes were visualized with anti-calreticulin (B) and anti-transferrin receptor (E) antibodies, respectively. Cell surface TrkA was revealed by immunolabeling of intact nonpermeabilized cells with an antibody directed against RTA (in H). Actin was stained with rhodamine-conjugated phalloidin (in K). Bars represent 10 µm.


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Fig. 3.   Cellular localization of TrkA-GFP chimera. Intracellular localization of TrkA was investigated in transiently transfected PC12 cells by confocal microscopy. Double labeling experiments were undertaken to analyze putative co-localization between TrkA-GFP construct (GFP fluorescence shown in green) and endosome (Tnf-R in red). Bars represent 10 µm.

                              
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Table I
Summary of co-localization analysis via confocal microscopy of the various TrkA-GFP constructs and markers of intracellular compartments
Cells were evaluated empirically for the levels of expression of the various constructs in the specified compartments identified through the use of specific markers described under "Materials and Methods." +/-, barely detectable; +, low expression; +++, highest expression; ND, not determined.

Synthesis, Maturation, and Cell Surface Translocation-- In PC12 cells, synthesis and cell surface translocation of TrkA involve the sequential production of two forms of the receptor. The receptor is synthesized initially in the rough endoplasmic reticulum as a 110-kDa form, which contains a 30-kDa N-linked sugar moiety (25, 26). During the time course of receptor targeting to the cell surface, this gp110TrkA precursor is matured to a 140-kDa form, gp140TrkA (Fig. 4A) The additional apparent 30-kDa molecular mass shift between mature and precursor forms of TrkA is believed to be the result of modification in the carbohydrate moiety of the receptor (54). Synthesis and maturation of TrkA-GFP chimerae were evaluated by Western blot (Fig. 4A). All of the constructs tested, with the exception of the soluble Delta TM mutant, exhibited two different molecular mass forms of the receptor. The molecular mass of the smallest forms appears to be about 30 kDa higher than the calculated molecular mass of the polypeptide backbone. This difference is similar to that observed for the endogenous TrkA of PC12 cells, suggesting that these extra 30 kDa correspond to the N-linked oligosaccharides normally added to the receptor. The molecular mass difference between the two forms of a chimeric receptor also appears to be close to 30 kDa, which is also comparable with the difference seen between gp110TrkA and gp140TrkA. These data suggest that synthesis and maturation of the TrkA-GFP chimera are similar to that observed with the wild type receptor. Pulse-chase analysis confirmed the precursor to mature protein relationship between the two forms of each chimeric receptor (not shown). It can be noted further from this experiment and from Western blot analysis that no preferential cleavage of GFP from the chimeric receptor was observed. These observations confirm the fidelity of GFP as a tag to follow the expression and fate of the TrkA receptor.


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Fig. 4.   Intracellular trafficking of TrkA-GFP fusion proteins. A, processing: Western blot analysis of total protein samples from PC12 nnr5 cells transiently transfected with TrkA-GFP constructs (Delta 0-Delta TM) or wt TrkA (TrkA). The left and right panels are probed with RTA antibody directed against TrkA extracellular domain and JL-8 antibody directed against GFP, respectively. B, cell surface targeting: Western blot analysis of total (T) versus cell surface (S) protein samples from PC12 nnr5 cells stably expressing TrkA-GFP constructs (see "Materials and Methods" for details). The left and right panels show blots probed with antibody directed against TrkA extracellular domain and GFP, respectively. C, NGF-induced internalization: PC12 nnr5 cells were transiently transfected with TrkA-GFP vector or wt TrkA along with a GFP expression vector. 48 h post-transfection, cells were collected and treated or not with 50 ng/ml NGF for 15 min. After being labeled with antibody against TrkA extracellular domain and phycoerythrin-conjugated secondary antibody, cells were used for flow cytometric analysis. Internalization efficiency was calculated on GFP-positive cells as described under "Materials and Methods." D, NGF-induced degradation: Western blot analysis of total protein sample from PC12 nnr5 cells transiently transfected with TrkA-GFP or wt TrkA constructs and treated or not with 50 ng/ml NGF for 5 h. Membranes were probed with antibody directed against the extracellular domain of TrkA.

Cell surface targeting of TrkA-GFP constructs was observed by both flow cytometric analysis and confocal immunofluorescence microscopy (Figs. 2 and 4C). Cell surface localization of TrkA-GFP chimerae was observed at the molecular level by cell surface biotinylation. In PC12 cells, the mature form of wild type TrkA is the only form translocated to the cell surface (Fig. 4B, left panel). As shown in Fig. 4B, Delta 0, Delta 1, Delta 2, Delta 4, and Delta 8 are also correctly targeted to the plasma membrane because only the mature forms of the chimeric receptors are labeled by biotinylation of cell surface proteins. Thus, neither exogenous receptor expression nor the addition of the GFP tag appear to affect maturation and cell surface targeting of TrkA in PC12 cells.

NGF-induced Down-regulation-- Intracellular trafficking of TrkA-GFP in response to NGF was analyzed. NGF binding to wild type TrkA induces internalization and degradation of the receptor. Flow cytometric analysis of NGF-induced internalization of TrkA-GFP chimerae from the cell surface was performed in transiently transfected PC12 nnr5 cells. Fig. 4C shows that the chimeric receptors can be separated into three categories with regard to NGF-induced internalization. The first category, which includes Delta 0 and Delta 1, demonstrated internalization properties similar to those of wild type TrkA. Delta 2, Delta 3, Delta 4, and Delta 6 showed slightly enhanced NGF-induced internalization as compared with the first group. Finally, the most deleted construct (Delta 8) has greatly reduced internalization capability. This observation suggests that there are at least two kinds of trafficking determinants in the cytoplasmic domain of TrkA, which regulate NGF-induced internalization. A negative regulator of this phenomenon is lost in the Delta 2-Delta 6 mutants, whereas a positive regulator of NGF-induced internalization is lost in Delta 8.

Following internalization of the NGF-TrkA complex, the receptor is targeted to the lysosome for degradation (11, 54). As shown in Fig. 4D, NGF-induced degradation of transiently transfected receptor is restricted to the Delta 0 and Delta 1 chimeric receptors and to wtTrkA. It suggests that the process of NGF-induced TrkA degradation requires trafficking information in the carboxyl terminus of the receptor, which is missing in the Delta 2-Delta 8 constructs.

Signaling Properties of the Chimerae-- We tested the ability of TrkA-GFP chimerae to promote signaling events upon binding of NGF. In PC12 cells, NGF treatment induces autophosphorylation of endogenous TrkA (Fig. 5A). PC12 nnr5 cells, a variant cell line devoid of TrkA receptor, do not respond to NGF (27, 28). Phosphorylation of TrkA-GFP chimerae was tested in PC12 nnr5 cells stably expressing TrkA-GFP constructs. As shown in Fig. 2A, NGF induces tyrosine phosphorylation of the mature form of Delta 0 and Delta 1 chimerae. As expected, the Delta 2 construct, which is lacking crucial amino acids in the catalytic domain of the kinase, is not phosphorylated in response to the growth factor. This observation suggests that the NGF-induced tyrosine phosphorylation of Delta 0 and Delta 1 is indeed the result of autophosphorylation of the receptor rather than a transphosphorylation of the chimera by an NGF-activated endogenous kinase.


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Fig. 5.   TrkA-GFP chimerae signaling. A, NGF-induced TrkA-GFP phosphorylation. PC12 nnr5 cells stably expressing TrkA-GFP constructs were treated for different periods of time with 50 ng/ml NGF or EGF. Cellular proteins were then extracted and immunoprecipitated with an antibody directed against phosphotyrosine (IP: alpha P-Y). Western blot (WB) analysis of total cellular extracts (C) or immunoprecipitated samples was then carried out with an antibody directed against TrkA extracellular domain or epidermal growth factor receptor (EGF-R) intracellular domain. B, cells were treated as described in A and subjected to Western blot analysis. Membranes were probed with an antibody directed against the phosphorylated active form of MAPK. C, PC12 nnr5 cells stably expressing Delta 0, Delta 1, or Delta 2 chimerae were treated or not for 2 days (2d) with 50 ng/ml NGF. Photomicrographs of cells were then acquired with phase contrast microscopy.

The kinetics of NGF-induced receptor autophosphorylation was also investigated. Fig. 5A shows that in PC12 cells, phosphorylation of endogenous TrkA in response to NGF is prolonged as compared with that observed for epidermal growth factor receptor in response to epidermal growth factor. The duration of NGF-induced phosphorylation of Delta 0 and Delta 1 also appears to be longer than that of epidermal growth factor receptor. Downstream of TrkA phosphorylation, activation of MAPK plays a key role in the response to NGF (29). Activation of these kinases is achieved by phosphorylation. Fig. 4B shows that NGF treatment of nnr5 cells stably expressing Delta 0 and Delta 1, but not Delta 2, induces phosphorylation and thus activation of MAPK.

The addition of NGF to PC12 cells promotes neuronal differentiation. PC12 nnr5 cells, devoid of endogenous TrkA, have lost this ability to differentiate in response to NGF (27, 28). Introduction of Delta 0 and Delta 1 into PC12 nnr5 cells restores NGF-induced differentiation, as shown in Fig. 4C. By contrast, the more deleted mutant, Delta 2, does not induce differentiation. These data indicate that the fusion of GFP to the carboxyl terminus of TrkA does not alter the signaling properties of the receptor. Moreover they show that deletion of the 11 carboxyl-terminal amino acids of the receptor, which include the phosphotyrosine involved in phospholipase C-gamma recruitment, does not affect NGF-induced morphological differentiation of PC12 cells (30).

Real-time Monitoring of Receptor Trafficking-- The trafficking of TrkA was followed using confocal time-lapse analysis of PC12 cells stably expressing Delta 0. The experiments presented in Fig. 5 represent a subset of data extracted from a single cell recording over a period of 5 days. Two representations are used in this figure. Black and white pictures represent recording at a single time point. Three-color pictures are compiled from the overlaying of three sequential recordings; the red channel is attributed to the first recording, the green channel to the second recording, and blue channel to the last recording. Thus, GFP labeling of structures that are immobile over the three recordings appears white (overlay of red, green, and blue channels), whereas successive positions of a moving structure appear as respectively colored marks.

Long-term Recording-- At the beginning of the NGF treatment, PC12 cells exhibit their undifferentiated round shape. In this cell, TrkA is found mainly at two locations. The bulk of the receptor appears to be concentrated in an internal perinuclear location. A part of the TrkA cellular pool is also found at or close to the plasma membrane. Receptors present at this site are accumulated in discrete locations of the cellular periphery, most often in the form of highly mobile cellular protrusions (Fig. 6, 18 h).


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Fig. 6.   Long-term confocal time-lapse analysis of TrkA-GFP trafficking. PC12 cells expressing Delta 0 chimera were grown on collagen-polylysine-coated plastic for 24 h. Live cells were then observed under a confocal microscope. Cells were treated with 50 ng/ml NGF, and pictures were recorded every 10 min, other than the period between 34 and 34 h 10 min, during which images were collected every 30 s. For explanations of colored versus black and white representations see the text.

After 21 h of NGF treatment, cells begin to enlarge and to extend neurites. During this process of neurite outgrowth, a large number of vesicles containing TrkA are transported to and from the plasma membrane. It can be noted that this flow of TrkA-containing structures to the cell surface at the beginning of neurite outgrowth is not a uniform process. Indeed TrkA-positive vesicles seem to be preferentially addressed to the plasma membrane locations from which neurite extension is attempted (Fig. 6, inset, 34 h). On the other hand, TrkA-positive membrane ruffles appear concentrated at the tip of potential neurites (inset, 34 h).

Neurite outgrowth appears to proceed by trial and error. As exemplified in the picture compiled from images taken from 47 h to 47 h 20 min (Fig. 6), some secondary neurites are retracted (arrow), whereas others appear to modify their direction of extension (asterisks). After 52 h of NGF treatment, neurites have retracted secondary outgrowths and extension is reduced. At this step of differentiation, TrkA localization and trafficking exhibit the following properties: First, TrkA is located in numerous internal fixed locations, moving from the initial perinuclear accumulation (Fig. 6, 52 h, red asterisk) to immobile structures in the proximal part of the neurite (Fig. 6, 52 h, green asterisks) and of the growth cone (Fig. 8D). Second, between these two latter locations, retro- and anterograde movements of vesicles (Fig. 8C) are detected. Third, the plasma membrane location of TrkA appears to be concentrated essentially in membrane ruffles at the growth cone and around the cell body (arrows in Fig. 6, 52 h).

After this stage of differentiation, the culture was treated with the TrkA inhibitor K252-a (0.5 µM). Treatment with this drug induces a dramatic change in cell morphology, and TrkA trafficking. K252-a induces rapid flattening of the cell body. This modification of cell shape is accompanied by a modification of membrane movement at the cell surface as shown by TrkA concentration in large yet discrete and highly mobile patches at the plasma membrane. Finally, TrkA undergoes a massive internal redistribution as assessed by anterograde vesicular transport from the proximal neurite "stations" to the perinuclear area (Fig. 7, inset at 72 h). After 7 h of drug treatment, a complete loss of peripheral TrkA-GFP is observed. Prolonged exposure to the drug led to apoptotic cell death (Fig. 7, 82 h 30 min).


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Fig. 7.   Long-term confocal time-lapse analysis of TrkA-GFP trafficking (continued from Fig. 6). PC12 cells expressing the Delta 0 chimera were grown on collagen-polylysine-coated plastic for 24 h. Live cells were then observed under a confocal microscope. Cells were treated with 50 ng/ml NGF, and pictures were recorded every 10 min for the first 52 h of differentiation (see Fig. 5). After 52 h of NGF treatment, the tyrosine kinase inhibitor K252-a was added to a concentration of 0.5 mM. Images were then acquired every 5 min.

Short-term Recording-- Long-term confocal time-lapse observations highlight the importance of vesicle shuttling between membrane ruffles and endosomal locations. Vesicle tracking studies were undertaken in HeLa cells. These cells are highly adherent and, as a consequence, are thinner than PC12 cells. These morphological properties of HeLa cells make them more suitable for analysis of vesicle tracking. Fig. 8A shows the overlay of three images recorded with a 1-s interval in HeLa cells stably expressing Delta 0. As seen in Fig. 8A, TrkA localization in this cell resembles the endosomal and cell surface location observed in PC12 cells. Immobilization of TrkA in these structures is revealed by the white labeling (Fig. 8A). By contrast, vesicles in transit between these two compartments undergo rapid movement (presence of colored vesicles). The multiplicity of vesicles going to and from the endosome does not allow single vesicle tracking. To overcome this problem, photobleaching of the endosome was performed (Fig. 8B). In this manner, fluorescence of Delta 0-positive vesicles emerging from endosomes is eliminated. Under such conditions, the tracking of vesicles moving from the cell periphery to the endosome is possible. The representative velocity of this endocytic, retrograde-like movement is 0.48 ± 0.07 µm/s (as evaluated from the measurement of 20 vesicles from six independent experiments).


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Fig. 8.   Short interframe confocal time-lapse analysis of TrkA-GFP trafficking. A and B, plasma membrane to endosome vesicle shuttling. HeLa cells expressing Delta 0 were cultured as described under "Materials and Methods." Confocal time-lapse analysis was undertaken with a 1-s interframe. The same cell is presented in A and B. In B, the internal endosomal Delta 0 fluorescence was photobleached, and NGF, 50 ng/ml, was added 5 min prior to the recording. C, vesicular transport in neurites. PC12nnr5 cells expressing Delta 0 were treated with NGF for 2 days at 50 ng/ml. Confocal time-lapse analysis was undertaken with a 1-s interframe. The cell body is located 200 µm to the left of the picture. The observed neurite divides in two parts. D, filopodial movement at the neurite tip. PC12nnr5 cells expressing Delta 0 were treated for 2 days with 50 ng/ml NGF. Confocal time-lapse analysis of a neurite tip was undertaken with a 7.5-s interframe. The cell body is located ~150 µm below the picture.

Recording of TrkA-GFP labeling during the time course of NGF-induced PC12 differentiation shows retrograde and anterograde vesicle movements in neurites. Recording over short time intervals in the cellular context of differentiated PC12 cells was also undertaken. The picture in Fig. 8C corresponds to the recording of a neurite emerging from a cell body, located to the left of the picture. This neurite divides in two parts in the middle of the picture. Nine seconds of tracking, at 1-s intervals, of a vesicle undergoing anterograde transport in this neurite is presented. The distance between the successive locations of the vesicle (red, green, and blue positions) is different in the three panels presented. In the first 3 s, the apparent vesicular velocity is about 0.26 µm/s. whereas it increases to about 0.4 µm/s and to about 1.3 µm/s in the next 6 s. This illustrates that linear vesicle velocity does not appear constant in the short-term recording experiments. Vesicle movement often appears irregular, with apparent pauses in the transport process. Moreover, despite the fact that global direction of the transport is maintained, it would appear that vesicles are undergoing rapid changes in transport orientation. The mean of vesicular TrkA-GFP anterograde transport was recorded at 0.46 ± 0.09 µm/s (as evaluated from measurement of 12 vesicles from four independent experiments).

TrkA-positive membrane ruffle mobility was evaluated in the long-term time-lapse recording experiment (Fig. 6). The images from the 72 to 72 h 10 min recording indicate that selected patches of plasma membrane, rich in TrkA, are moved around the cell surface in a concerted manner. It is conceivable that in the context of a differentiated cell, retrograde transport of the receptor may occur via such membrane ruffles. This hypothesis is supported by the results presented in Fig. 8D. Observation of the growth cone reveals that membrane ruffles move around the surface. Asterisks show the successive positions of a ruffle for which the velocity of displacement, in this particular experiment, is ~0.3 µm/s.

Trafficking and Activation-- Confocal time-lapse analysis indicated numerous bidirectional movements of TrkA-containing vesicles between the cell surface and the perinuclear endosomal compartment (Figs. 6-8). Experiments were designed to evaluate the activation state of TrkA receptors involved in these movements. Activation status, together with ligand-binding status of TrkA, was evaluated using confocal microscopy (Fig. 9; and see "Materials and Methods" for details). Expression of chimeric TrkA-EGFP receptor in PC12 nnr5 cells together with the use of RTA antibody as an NGF agonist (22) allow simultaneous monitoring of three different pools of cellular TrkA in a single cell (Fig. 9A) as follows. (i) "Total TrkA" is reflected in GFP fluorescence, which corresponds to all of the cellular localizations of TrkA. (ii) "Cell Surface TrkA at t = 0" is identified by incubation of cells at 4 °C for 30 min in the presence of RTA, followed by permeabilization and labeling of cells with rhodamine-conjugated anti-rabbit antibody. This allows the detection of TrkA-RTA complexes, which are initially at the cell surface. Following the shift to 37 °C, it is possible to monitor the fate of the receptors that have moved from the cell surface to intracellular locations during the incubation time. (iii) "Activated TrkA" corresponds to immunolabeling of permeabilized cells using anti-phospho-TrkA specific antibody.


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Fig. 9.   Trafficking and activation of TrkA-GFP chimerae. Internalization of the TrkA-GFP chimerae was provoked by exposure of PC12 cells to RTA as described under "Materials and Methods." After fixation and permeabilization of the cell, RTA localization was evaluated by exposure to secondary antibody labeled with rhodamine at the indicated times. Thus, rhodamine fluorescence indicates the location of TrkA-RTA complexes that were at the cell surface at the beginning of the experiment (Cell Surface TrkA at t = 0 min). GFP fluorescence reflects all cellular TrkA (Total TrkA). Simultaneous immunolabeling with anti-phospho-TrkA antibody (A) or anti-transferrin antibody (B) allows detection of "Activated TrkA" or the "Endosome," respectively.

As shown in Fig. 9A, incubation of cells in the presence of RTA at 4 °C does not allow receptor internalization, because all RTA-TrkA complexes are located at the cell surface. Moreover, incubation at 4 °C does not permit TrkA activation, because no phospho-TrkA is detected at this temperature. By contrast, incubation of cells for 20 min at 37 °C in the presence of RTA led to the accumulation of TrkA-antibody complex in discrete structures within cells. Intracellular RTA-TrkA complexes are found both at the cell periphery (arrows) and in the aforementioned perinuclear location (arrowheads). RTA-TrkA complexes located at this latter location also appear to correspond to phosphorylated receptor. It can be noted that activated receptor is detected in a perinuclear area that is larger than the perinuclear location of internalized RTA-TrkA complexes. Fig. 9B illustrates that internalized TrkA is targeted to endosomes, because the perinuclear RTA-TrkA complexes appear to co-localize with the endosomal marker, Tnf-R.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study validates the use of TrkA-GFP chimerae as a tool to follow intracellular trafficking of TrkA. Data presented herein indicate that wt TrkA-GFP (Delta 0) fulfills most of the trafficking and signaling properties of wt TrkA. Analysis of receptor location in the absence of NGF shows that this transmembrane receptor is accumulated in specific areas of the plasma membrane and in endosomes within the cell. Monitoring of chimeric receptor trafficking in live cells sheds new light on the biosynthetic and endocytic trafficking pathways followed by TrkA. Observation of truncated TrkA-GFP constructs permitted identification of those parts of the receptor intracellular domain that are necessary for different steps of receptor trafficking. Receptor maturation and cell surface translocation are not affected by the removal of the entire TrkA cytoplasmic domain. Intracellular distribution of the receptor is modified by deletion of amino acids 788-793. All chimerae encompassing this deletion exhibit a reduction in the amount of receptor that is retained intracellularly in the absence or presence of NGF. Down-regulation events induced by NGF also appear to be modulated by sequences within the intracellular domain.

The intracellular domain of TrkA contains a tyrosine kinase, which is flanked by short juxtamembrane and carboxyl-terminal domains, each including a single tyrosine known to be involved in signal transduction (4, 31). Fusion of the 26-kDa GFP protein to the carboxyl terminus of the 40-kDa intracellular domain of TrkA does not perturb the cellular and biochemical properties of the receptor as compared with wt TrkA. The Delta 0 chimera is activated in response to NGF, leading to MAPK activation and differentiation comparable with that observed with the wt receptor. The activation pattern of TrkA is not modified by the addition of GFP, because Delta 0 exhibits activation kinetics similar to that of TrkA. This latter point is believed crucial for the cellular response to tyrosine kinase activation in PC12 cells. Indeed proliferative versus differentiative responses induced, respectively, by EGF and NGF are correlated with transient versus sustained signaling by their respective tyrosine kinase receptor (32). Data presented herein indicate that Delta 0 also shows a sustained activation similar to that of wt TrkA.

Numerous pathways for signal transduction have been described for NGF signaling in PC12 cells, many of which are initiated from TrkA (30, 33, 34). Clearly, short of performing a catalogue of each, it is not possible to ensure that all are fully functional in the TrkA-GFP chimera. However, the PC12 nnr5 model, devoid of functional TrkA, allows the evaluation of the global cellular response as reflected by the morphological differentiation accompanied by the anti-mitogenic response to NGF, which are observed subsequent to expression of the Delta 0 and Delta 1 chimera.

Delta 0 trafficking appears similar to that of wt TrkA. The transit of Delta 0 through the biosynthetic pathway allows correct receptor maturation and cell surface translocation. Moreover, NGF-induced down-regulation of Delta 0, namely internalization and degradation, mimic that observed for wt TrkA. Taken together, these data suggest that TrkA-GFP mimics wt TrkA behavior.

The presence of the GFP on the carboxyl terminus of TrkA does not modify the turnover of this protein. Furthermore, both Western blot analysis and pulse-chase experiments failed to detect any preferential cleavage of GFP from the chimera. Thus, GFP appears to be a valuable marker to follow expression and fate of TrkA in cells.

In the absence of NGF, Delta 0 is distributed between actin-rich cell surface membrane ruffles and internal endosomal locations. Confocal time-lapse analysis allows the monitoring of trafficking of the receptor between these structures. During the process of differentiation, TrkA-positive membrane ruffles appear to be concentrated at the growth cone and around the cell body. It has been shown that actin is involved in the retrograde transport of NGF (35). In light of these studies, this requirement may rely on two kinds of retrograde transport processes. First, actin may participate in an internalization-dependent retrograde transport of TrkA. The observation that NGF treatment causes membrane protrusion (35) and the present observation that TrkA is specifically enriched in these plasma membrane areas raise the possibility that TrkA internalization could be achieved by macropinocytosis. Indeed, this internalization process, as contrasted with coated pit-mediated endocytosis, does not require membrane invagination but membrane protrusion (36, 37). Alternatively, actin may be required in a retrograde transport mechanism of TrkA, which involves membrane ruffle transport (38). The localization of TrkA in the membrane ruffle at the growth cone is also consistent with its involvement in neurite tip oriented growth. Indeed, ruffle movements at the growth cone are believed to "taste" extracellular medium (39). Local TrkA signaling in ruffles could support this function of the receptor.

The internal accumulation of neurotrophin receptors has been described previously for both TrkA (24, 40) and TrkB (41). The observed accumulation of TrkA-GFP in an endosomal compartment in the absence of NGF treatment is consistent with these observations. Bidirectional vesicular receptor transport between endosomal structures and the cell surface indicates a highly dynamic equilibrium within these two receptor locations. Calculation of the average velocity of anterograde and retrograde vesicle transport (respectively 0.46 ± 0.09 µm/s and 0.48 ± 0.07 µm/s) suggests that the vesicular transport mechanism is of the fast axonal type (42). These values are in agreement with the in vivo retrograde transport velocity of NGF (from 0.7 to 0.8 µm/s (43, 44)).

Confocal time-lapse analysis highlights the fact that a significant proportion of TrkA is located intracellularly at the level of endosomes, even in the absence of growth factor, in agreement with published results concerning the expression of endogenous TrkA (24). The availability of antibodies specific to the extracellular domain of rat TrkA, with a well characterized NGF agonist function, allows monitoring of ligand-receptor complex movement from the cell surface (22). This analysis shows that internalized TrkA is indeed targeted to the endosome, as suggested by several groups (16, 17, 45). The studies presented herein take these observations even further and clearly illustrate that the internalized, activated TrkA is located mainly in endosomes (54). A striking finding of this analysis is the fact that activated TrkA is present in areas of the endosome devoid of the internalized receptor-ligand complexes. This suggests that internalized receptor may serve to propagate activation from the cell surface to the fraction of TrkA accumulated in the endosome, thereby serving to amplify the intracellular signal even though these receptors are not occupied with ligand. This concept has been discussed by numerous investigators in the context of neurotrophic factor signaling from the nerve ending to the cell body and is at the heart of lively debate (14, 46-48, 52). It is expected that molecular analysis of XFP-labeled neurotrophic factor receptors, coupled with the multi-label time-lapse and confocal microscopy approaches described here, could facilitate the clarification of this and other questions concerning receptor trafficking and signal transduction.

In the context of differentiated PC12 cells, internal distribution of the receptor appears not to be restricted to the perinuclear endosomal location. For example, TrkA-GFP-positive structures have been observed in the neurite tip. Hendry and colleagues (49, 50) have recently proposed the existence of a sorting compartment located in the growth cone responsible for the targeting of internalized NGF to retrograde transport. The observed internal accumulation of TrkA-GFP inside the neurite tip could represent this sorting station. This hypothesis is strengthened by the observed bidirectional vesicular transport between both the neurite tip cell surface and internal structures and between these latter locations and the cell body.

TrkA targeting into NGF-dependent and -independent trafficking pathways appears to rely on different regions of the receptor cytoplasmic domain. Maturation and cell surface targeting of TrkA are apparently not regulated via motifs within cytoplasmic domain. Anchoring of the receptor in the membrane appears to be the only requirement for correct maturation of the receptor ectodomain. As mentioned above, at steady state, in the presence or absence of NGF, TrkA-GFP is accumulated in the endosome, yielding a strong fluorescence. It would appear therefore that activation of the tyrosine kinase is not required for TrkA to traffic to this endosomal location. However, the amount of TrkA-GFP present in the endosome is significantly decreased when amino acids 788-673 are deleted. Again, NGF has no effect on this distribution. These forms of TrkA are kinase-dead because of the absence of the activation loop in the kinase domain. By contrast, inactivation of kinase activity via a single substitution in the TrkA ATP binding site (Delta 0-KD chimera) does not affect endosomal localization. Together, these results would suggest that the difference in localization between these deletion mutants and the full-length construct is due to the absence of a trafficking motif such as YRKF rather than the lack of kinase activity.

NGF-induced trafficking steps include receptor internalization followed by receptor degradation. Tyrosine kinase activity of TrkA does not appear to be necessary for NGF-induced internalization. Receptor internalization is actually enhanced for the kinase-dead truncated constructs Delta 2, Delta 3, Delta 4, and Delta 6. This observation suggests that the tyrosine kinase activity domain of the receptor may be a negative regulator of NGF-induced internalization. It also suggests that the mechanism of internalization does not require conformational modification of the receptor cytoplasmic domain, believed to be achieved by tyrosine kinase activation. Thus, NGF-induced internalization may be triggered by NGF-mediated TrkA dimerization. With regard to this hypothesis, adaptor protein binding to trafficking signals in the coated pit pathway has been shown to be enhanced by dimerization of the internalized protein (51). Further deletion of amino acids 565-477 in the cytoplasmic domain of TrkA (Delta 8 mutant) greatly reduces NGF-induced internalization. Thus, a positive regulator of NGF-induced internalization, such as the putative trafficking motifs 531ECYNLL or 496NPQY, is probably present in this portion of the TrkA cytoplasmic domain. A tempting hypothesis to link these two types of internalization regulators is the competitive binding of signaling and trafficking effectors on a single motif of the TrkA cytoplasmic domain. Activation of the tyrosine kinase by NGF may inhibit internalization of the receptor by favoring the recruitment of signaling rather than trafficking effectors on the phosphorylated 496NPQY motif. With regard to this hypothesis, abolishing receptor tyrosine kinase activity would invert the signaling/trafficking binding balance on the motif, and deletion of the motif would abolish the binding of both effector types.

The reagents and observations presented herein should offer a powerful means for investigating the relation between trafficking of and signaling via TrkA as well as other receptors.

    ACKNOWLEDGEMENT

We are extremely grateful to Dr. Pierre Colas of our group (Lyon, France) for advice in molecular biology.

    FOOTNOTES

* This work was supported by grants from the Ligue Nationale Contre le Cancer, the committee of the Ligue from the Rhône, the Rhône-Alpes Region, the Association for Research against Cancer (ARC), and the Fondation de France.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by grants from the Ministère de l'Enseignement Supérieur et de la Recherche and fellowships from the Association for Research against Cancer.

** An investigator of the Howard Hughes Medical Institute; work in his laboratory was also supported by United States Public Health Service Grant NS 16033.

Dagger Dagger To whom correspondence should be addressed. Tel.: 334-72-72-81-96; Fax: 334-72-72-80-80; E-mail: bbrudkin@ens-lyon.fr.

Published, JBC Papers in Press, November 15, 2002, DOI 10.1074/jbc.M202401200

    ABBREVIATIONS

The abbreviations used are: NGF, nerve growth factor; MAPK, mitogen-activated protein kinase; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; PBS, phosphate-buffered saline; RTA, antibody against extracellular domain of TrkA; wt, wild type; Delta TM, transmembrane domain deletion construct; Delta ECT, extracellular domain deletion construct.

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ABSTRACT
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RESULTS
DISCUSSION
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