Article |
Address correspondence to Simon Halegoua, Dept. of Neurobiology and Behavior, Life Sciences Bldg. 052, SUNY at Stony Brook, Stony Brook, NY 11794-5230. Tel.: (631) 632-8736. Fax: (631) 632-9714. E-mail: simon.halegoua{at}sunysb.edu
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Abstract |
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Key Words: neutrophin; membrane trafficking; signal transduction; EH domain; MAP kinase
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Introduction |
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Although retrograde transport of NGF would appear to be important for NGF actions, the mechanisms for NGF internalization, formation of the transport vehicle, and its interaction with the transport machinery have been elusive. NGF is internalized after binding to its plasma membrane receptors, p75NTR, and TrkA. NGF and TrkA have been reported to be associated with and/or stimulate different types of endocytic machinery in PC12 cells, including clathrin-coated vesicles (Grimes et al., 1996; Howe et al., 2001), ruffling and pinocytosis (Connolly et al., 1987), and calveolae (Huang et al., 1999). The means by which NGF and TrkA are internalized may involve any combination of these processes. As opposed to the well-defined modes for processing receptor-mediated endosomes (for review see Mellman, 1996), a remarkable feature of NGF-containing endosomes in neuronal terminals is that they can avoid degradation or recycling, and instead can be directed to the retrograde transport machinery.
How does the NGF received at the neuronal terminal mediate signaling at the cell body? It has been proposed that NGF filled endosomes with the capacity for receptor-mediated intracellular signal transduction function during and after retrograde transport (Halegoua et al., 1991). Putative signaling endosomes containing NGF and TrkA have since been identified and isolated from PC12 cells (Grimes et al., 1996). Although signaling pathways activated by retrogradely transported NGF and TrkA have only recently been investigated, TrkA signaling at the plasma membrane is well documented, and is mediated through multiple pathways initiated by specific TrkA autophosphorylation sites (for review see Kaplan and Miller, 2000). The two best-studied signaling sites on TrkA, P-Y490 and P-Y785, bind to Shc and phospholipase C- (PLC-
), respectively. Shc mediates stimulation of Ras-mitogen -activated protein (MAP) kinase and PI-3-kinase signaling pathways, whereas PLC-
mediates signaling via phosphatidylinositol turnover. These signaling pathways are further branched, leading to differential control of gene expression (D'Arcangelo and Halegoua, 1993) and survival (for review see Kaplan and Miller, 2000). Retrograde axonally transported TrkA has also been shown to be autophosphorylated (Ehlers et al., 1995; Riccio et al., 1997) on the Shc-binding site Y490 (Bhattacharyya et al., 1997), and mediates CREB phosphorylation in the cell body (Riccio et al., 1997; Watson et al., 1999), which is in part necessary for neuronal survival (Riccio et al., 1999). Several studies have suggested that internalized TrkA may signal differently from the plasma membrane receptor, although a consensus on the different signaling parameters has not yet been reached (Saragovi et al., 1998; Zhang et al., 2000; Wu et al., 2001). A recent study has identified the erk5 MAP kinase as selectively stimulated in the cell body by retrogradely transported TrkA (Watson et al., 2001).
Understanding the mechanisms for the formation and processing of, and signaling from, the endosomal NGF/TrkA complex, have been hampered by a lack of tools with which to specifically manipulate internalization of the NGF/TrkA complex and generation of the signaling endosome. We have identified a new NGF-induced protein in PC12 cells termed Pincher that functions as a pinocytic chaperone for vesicles containing NGF and TrkA. Pincher function is necessary for both the NGF-induced internalization of TrkA by a pinocytic process, and the sorting of long-lived endosomal vesicles with NGF signaling capability. Pincher function may shed light on the process of retrograde endosomal NGF signaling.
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Results |
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A full-length cDNA for HAL18 was created from overlapping PC12 library cDNAs and the predicted open reading frame is shown in Fig. 1 C. The only difference noted between clones was the length of 5' untranslated sequence, suggesting that the two mRNAs detected on Northern blots represent alternate splice forms. For reasons discussed below, this protein was called Pincher. An antibody raised to a glutathione-S-transferase (GST)Pincher fusion protein immunoprecipitated a protein from PC12 cell extracts that was detected on Western blots probed with the same antibody (Fig. 1 D). No Pincher protein could be detected in blots prepared from immunoprecipitates using anti-Pincher antibody that was preblocked with GSTPincher (unpublished data). Pincher was induced by NGF treatment of PC12 cells over a time course that corresponded in magnitude and followed the time of induction of the HAL18 mRNAs detected on Northern blots. The tissue distribution of Pincher expression was determined by probing Northern blots containing RNAs from various tissues. Expression of both Pincher mRNAs was observed in heart and lung, dorsal root ganglion peripheral neurons, kidney, and brain (Fig. 1 E). Protein was also detected immunocytochemically in various neuronal populations in brain and in sympathetic neurons (unpublished data).
The sequence of Pincher revealed that it was a member of a gene family containing four mammalian genes (human EHD-1 to EHD-4 and mouse mRme-1 genes) (Pohl et al., 2000; Lin et al, 2001) and a nematode gene (RME-1) (Grant et al., 2001). Pincher-coding cDNA sequence is most closely related to the human EHD-4 gene (90% nucleic acid identity) (Kuo et al., 2001). Analysis of the predicted Pincher amino acid sequence revealed several interesting domains (Fig. 1 C), including an EH membrane trafficking domain, a coiled-coiled domain, and a domain distantly related to AAA type ATPases containing an intact P-loop ATP/GTP-binding motif. Because of these structural features, and its induction by NGF, we decided to examine whether Pincher was involved in TrkA trafficking.
Pincher overexpression enhances NGF-induced TrkA endocytosis
To determine the localization and effects of Pincher on the distribution of TrkA in PC12 cells, TrkA-PC12 cells that overexpress TrkA were transfected with a mammalian expression vector encoding a hemagglutinin (HA)-tagged Pincher. HA-Pincher expression was confirmed in Western blots of cell extracts from transfected cultures (Fig. 1 D). HA-Pincher localization was examined by confocal immunofluorescence microscopy using anti-HA antibody. As seen in Fig. 2 A, HA-Pincher is localized primarily to the plasma membrane, but can be occasionally visualized intracellularly. Plasma membrane localization was confirmed by costaining with wheat germ agglutinin, which showed a greatly overlapping staining pattern (unpublished data). The distribution of HA-Pincher did not appear to depend on the level of HA-Pincher expression as indicated by the similar pattern of staining for both dimly and brightly stained cells. TrkA, visualized in the same cells by double labeling using anti-TrkA antibody, was normally localized to the plasma membrane in a pattern that partially overlapped with that of HA-Pincher (Fig. 2 A). The small level of intracellular TrkA staining was presumably associated with the endoplasmic reticulum in the TrkA-PC12 cells. After NGF treatment, both HA-Pincher and TrkA migrated away from the peripheral plasma membrane in a time-dependent manner. After 2 min of NGF treatment, both HA-Pincher and TrkA became preferentially associated with NGF-induced ruffles and blebs at the plasma membrane (Fig. 2 A). Within 3 min of NGF treatment, cells were also seen in which HA-Pincher and TrkA were both dramatically associated with intracellular vesicle-like structures in largely overlapping patterns (Fig. 2 A). Although NGF treatment rapidly caused an apparent colocalization and internalization of HA-Pincher and TrkA, within 10 min of NGF treatment HA-Pincher and TrkA localization became differentially reorganized. HA-Pincher was associated predominantly with an unusual array of intracellular structures (Fig. 2 A, NGF 10 min). The Pincher-containing arrays were sorted away from a centralized collection of TrkA-containing structures. Pincher arrays were often found surrounding the TrkA-containing structures, as shown in the three-dimensional reconstruction of the cell in Fig. 2 C. Within 1 h of NGF-treatment, HA-Pincher was again found predominantly at plasma membrane and juxtamembrane locations, but rarely in internal structures, whereas TrkA was observed to have accumulated mainly in a dense collection of intracellular vesicle-like structures (Fig. 2 A), which were never seen to be associated with the nucleus. In many cells, as depicted in Fig. 2 A, TrkA was extensively internalized and was barely detected at the plasma membrane.
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Pincher-generated vesicles mediate NGF/TrkA signaling
The above results indicated that NGF was internalized with TrkA; thus, we expected that the internalized TrkA might remain activated and autophosphorylated (Bhattacharyya et al., 1997). To test this possibility, we used an antiphospho-Y490TrkA antibody in confocal immunofluorescence microscopy of Pincher-transfected, TrkA-PC12 cell cultures. As expected, before NGF treatment, Pincher- overexpressing cells detected with anti-HA antibody did not stain well with antiphospho-Y490TrkA antibody (Fig. 8 A). However, after NGF treatment, a time-dependent pattern of antiphospho-Y490TrkA staining was observed that was remarkably similar to that described above using anti-TrkA antibody. Within 2 min of NGF treatment, antiphospho-Y490TrkA staining could be seen at the plasma membrane, concentrated together with Pincher at membrane ruffles and blebs (Fig. 8 A). Pincher overexpression was found to increase both the appearance of phospho-TrkA in ruffles after NGF treatment (Fig. 8 A), and the level of TrkA autophosphorylation. Staining of phosphorylated TrkA was seen in 67% of the Pincher-overexpressing cells (n = 70) compared with 28% (n = 100) of the cells not overexpressing Pincher, when assayed at 10 min of NGF treatment. Between 5 and 15 min of NGF treatment, a massive internalization of both HA-Pincher and phospho-Y490TrkA was seen accumulated in the cytoplasm in both overlapping and nonoverlapping patterns. As seen with TrkA staining described above, at 15 min of NGF treatment, cells could be seen in which the accumulation of internalized phospho-TrkA was surrounded by HA-Pincher-labeled tubule-like structures (Fig. 8 A). Cytoplasmic staining of internalized phospho-Y490TrkA was seen in 89% of those Pincher-overexpressing cells, compared with 34% of the phospho-TrkA stained cells not overexpressing Pincher. In addition, the extent of internalization per positive cell appeared dramatically higher in the Pincher-overexpressing cells, compared with untransfected cells (Fig. 8 A). After 1 h and for as long as 24 h of NGF treatment, Pincher was found predominantly on the cell surface, whereas phospho-Y490-TrkA staining was bright and in a concentrated array of cytoplasmic vesicles in a distribution that was not seen in untransfected cells (Fig. 8 A), as was seen for TrkA and myc-NGF. In many cells, as shown in Fig. 8 A, little phosphorylated TrkA was seen at the cell surface, suggesting that the internalization was sufficient to significantly deplete activated TrkA from the cell surface.
The persistence of TrkA autophosphorylation in intracellular vesicles suggested that they would have the capacity to mediate pertinent downstream signaling events in the cytoplasm. To test this possibility, we examined the distribution of NGF-activated MAP kinases, whose activation is mediated through phospho-Y490 TrkA signaling. Phosphorylation of the erk MAP kinases is persistently stimulated in PC12 cells, and erk5 kinase is selectively activated in neuronal soma by retrograde axonal NGF signaling (Watson et al., 2001). Activated erk5 was examined using an antiphospho-erk5 kinase antibody in immunofluorescence staining of TrkA-PC12 cells transfected with a Pincher expression plasmid. Before NGF treatment, Pincher overexpressing cells detected with anti-HA antibody, showed low level staining with the antiphospho-erk5 kinase antibody (Fig. 8 B). However, after NGF treatment, a time-dependent pattern of phospho-erk5 kinase staining was observed that was remarkably similar to that described above using the antiphospho-Y490TrkA antibody. Within 5 min of NGF-treatment, bright phospho-erk5 kinase staining could be seen at the plasma membrane concentrated with Pincher, and colocalized with Pincher upon internalization (Fig. 8 B). By 15 min of NGF-treatment, phospho-erk5 kinase staining was seen in large concentrated punctate formations in the cytoplasm. As was seen with phospho-TrkA staining, the bright cytoplasmic staining of phospho-erk5 kinase was often surrounded by the Pincher arrays (Fig. 8 B). Within 1 h and for as long as 24 h of NGF treatment, Pincher was again seen associated predominantly with the plasma membrane, whereas phospho-erk5 kinase remained concentrated in the cytoplasm (Fig. 8 B). As exemplified in Fig. 8 B and similar to the distribution of phospho-TrkA, although bright staining of phospho-erk5 kinase was seen in a diffuse punctate pattern throughout the cytoplasm of nontransfected cells, the patterns of concentrated formations of phospho-erk5 kinase were only seen in Pincher-overexpressing cells. To determine if the distribution of phospho-erk5 overlapped with that of TrkA, Pincher-transfected cells were double labeled with monoclonal anti-TrkA and polyclonal antiphospho-erk5 antibodies. As shown in Fig. 8 C, the intracellular staining patterns of TrkA and phospho-erk5 largely overlapped at both 10 and 60 min NGF treatments. Unlike the punctate distribution of phospho-erk5, the pattern of phospho-erk1/2 kinase staining after NGF treatment of PC12 cells, as recently reported (Wu et al., 2001), was diffusely distributed throughout the cytoplasm and nucleus (Fig. 9 A). A similarly diffuse pattern of phospho-erk1/2 staining was generally seen in HA-Pincheroverexpressing cells at all times of NGF treatment (unpublished data). At 24 h of NGF treatment, when the greatest number of cells showed large aggregates of cytoplasmic phospho-TrkA staining, 79% (n = 33) of the antiphospho-erk5stained cells showed similar large aggregate staining patterns (Fig. 8 B), whereas only 15% (n = 68) of cells showed a similar pattern with antiphospho-erk1/2 staining, with the vast majority (85%) of the cells demonstrating diffuse phospho-erk1/2 staining (unpublished data).
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The distribution of activated erk5, seen by antiphospho-erk5 staining, indicated that like phospho-TrkA, phospho-erk5 remained at or very near the plasma membrane during NGF treatment of PincherG68E expressing cells (Fig. 9 A). This contrasts with the NGF-induced cytoplasmic staining pattern of phospho-erk5 normally seen in untransfected cells (Figs. 8 B and 9 A), or in cells overexpressing Pincher (Fig. 8, B and C), which largely colocalizes with TrkA (Figs. 8 C and 9 A). As expected, in PincherG68E-expressing cells, double labeling with both TrkA and phospho-erk5, indicated greatly overlapping patterns (Fig. 9 A). The pattern of phospho-erk5 staining differed from that of phospho-TrkA in that intracellular phospho-erk5 staining was never seen in cells overexpressing PincherG68E. In contrast to the pattern of phospho-erk5, phospho-erk1/2 displayed a normal distribution pattern after NGF treatment of cells expressing PincherG68E. Phospho-erk1/2 staining was bright and diffuse throughout the cytoplasm and nucleus after 15 min of NGF treatment (Fig. 9 A), and at all later times examined. Thus, the expression of Pincher G68E selectively prevented cytoplasmic signaling by erk5, but not by erk1/2.
To determine if clathrin-mediated endocytosis still occurred in cells expressing PincherG68E, the internalization of transferrin was examined in these cells. As shown in Fig. 9 B, PincherG68E did not change its plasma membrane localization upon treatment of cells with transferrin, and its expression had no effect on the extent or pattern of transferrin internalization.
The above results suggested that expression of PincherG68E blocked NGF-induced endocytosis at an early step. To gain insights into the stage at which internalization was blocked, we examined the distribution of PincherG68E by immunogold electron microscopy of NGF-treated cells. As shown in Fig. 10, although PincherG68E was found only at blebs at the cell surface, no complex ruffling structures were seen that were indicative of macropinocytic endocytosis. However, examples could be seen of clathrin-coated pits formed at the cell surface (Fig. 10).
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Discussion |
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The regulation of gene expression by NGF is mediated through multiple signaling pathways derived from TrkA. NGF-induced gene expression changes are mediated through TrkA by either triggered PLC- signaling (Choi et al., 2001), or by Shc- and Ras-mediated signaling that requires persistent stimulation (Segal and Greenberg, 1996). Unlike other genes thus far studied, Pincher induction by NGF is mediated through a combination of these TrkA-stimulated pathways. Mutation of the TrkA binding sites for PLC-
or for Shc resulted in partial inhibition of Pincher induction, whereas mutation of both sites completely blocked induction of Pincher by NGF. This is much like the paradigm seen with NGF-induced neurite growth that can be stimulated through both PLC-
and Shc binding sites on TrkA (Stephens et al., 1994), a mechanism that has been suggested to ensure the elicitation of important events by NGF. Increased Pincher production thus might ensure continued and perhaps even increased endosomal signaling with persistent NGF exposure.
Pincher facilitates the internalization and sorting of intracellular vesicles containing NGF and TrkA. Pincher would appear to function early in the internalization process because it was found primarily on the plasma membrane and was rapidly distributed, together with NGF and TrkA, to sites of membrane ruffling and pinocytosis after NGF treatment. The massive array of pinocytic structures at ruffling membrane blebs was found only in NGF-treated cells overexpressing Pincher, suggesting that Pincher drives the macropinocytotic process. Because Pincher-stimulated TrkA internalization required NGF-treatment, and ATP/GTP binding site (P-loop) mutants of Pincher blocked NGF-induced receptor internalization, it is likely that Pincher is a mediator of NGF-induced pinocytic internalization of TrkA. The time course of Pincher localization offered insights into the stages of the internalization process. The large complexes of pinocytic ruffling structures were internalized en mass, and developed into Pincher-containing tubular structures, concomitant with the appearance of vesicles within the tubules. In key respects the Pincher pinocytic process resembled receptor-induced macropinocytosis (Swanson and Watts, 1995) in that Pincher was enriched in NGF-induced ruffling membrane blebs where pinocytosis occurred, and the pinocytic vesicles were filled with extracellular fluid, as evidenced by the inclusion of media-soluble dextran. Although the appearance of large masses of pinocytic structures was atypical, it may reflect an unusually high degree of coupling of pinocytosis to ruffles caused by Pincher overexpression. Interestingly, TrkA was concentrated at NGF-induced ruffles and blebs of the plasma membrane, which resulted in an enrichment of TrkA in the internalized membranes. The specificity for receptor-enriched membrane internalization provided by the Pincher-driven internalization of TrkA, suggests a unique mode of receptor-mediated pinocytic endocytosis.
A late stage of Pincher-stimulated pinocytic endocytosis is the sorting of vesicles containing NGF and TrkA from the tubules, and recycling of the Pincher-containing tubules to the plasma membrane. This process was dramatically enhanced in Pincher-overexpressing cells, allowing visualization of intermediates that have not been described previously. A surprising finding was the presence of vesicles within many of the Pincher-containing tubules and the connections of tubules to vesicle accumulating bodies. The tubules may represent endosome/pinosome processing centers providing a unique generating, sorting and delivery system for endosomal vesicles to the cell interior. It is not clear at present how the vesicles are formed within the tubules, or how and when TrkA is sorted differentially from the Pincher-containing membrane. Pincher recycling to the plasma membrane occurs via recycling of Pincher-containing tubules. After delivery of NGF/TrkA vesicles to vesicle accumulating bodies, the tubules become EM lucent and could at these late stages be seen to be contiguous with the plasma membrane. The NGF/TrkA vesicles that are accumulated in the cytoplasm persist for periods of as long as 24 h, indicating that the process is distinct from endocytic events associated with lysosomal degradation or receptor recycling. In concordance with this idea, we also did not observe significant co-staining of Pincher with either clathrin or with the late endosomal marker LBPA at any times after NGF treatment (unpublished data). Pincher-mediated NGF and TrkA internalization appear to be mediated through a clathrin independent process. The differential effects of Pincher in stimulating, and PincherG68E in blocking, TrkA versus clathrin-mediated transferrin internalization best illustrate this. Consistent with these results, Pincher does not associate with and PincherG68E does not prevent, clathrin coated pits seen electron microscopically. However, the decrease in clathrin-mediated transferrin uptake in Pincher-overexpressing cells may be an indication that the Pincher and clathrin endocytotic machineries might share common components. That the unique mode of Pincher-mediated pinocytic endocytosis may provide for the formation and segregation of vesicles with long lifetimes is an intriguing possibility.
Recent reports have implicated Pincher family members in events underlying receptor-mediated endocytosis. The mouse mRME-1 gene product has been suggested to mediate recycling of receptor-containing endocytic vesicles (Lin et al., 2001). This function is unlike that for Pincher, which did not mediate recycling of the TrkA-containing vesicles that persisted in the cytoplasm. Furthermore, mRME-1 overexpressed in TrkA-PC12 cells did not act like Pincher to enhance NGF-induced TrkA internalization (unpublished data). Also, unlike Pincher, EHD-1 was recently suggested to be involved in clathrin-mediated endocytosis of IGF-1 (Rotem-Yehudar et al., 2001). The differences between Pincher and mRME-1 or EHD-1 suggest that the different family members may have different functions in vesicle transport. It is tempting to speculate that a common functionality of Pincher and other family members lies in the ability to pinch membrane, which in the case of mRME-1 results in the formation of recycling vesicles and in the case of Pincher results in macropinocytotic structures. The tissue distribution of Pincher mRNA is similar to that of mRME-1 and EHD-1, with high levels of expression in heart and lung, suggesting that Pincher may also be involved in a broader spectrum of receptor-mediated pinocytic events. The ability of Pincher to mediate internalization of receptors for neurotrophins and growth factors other than NGF remains to be determined.
The intracellular vesicles that accumulated after NGF-treatment of Pincher-overexpressing cells had signaling capability for NGF, similar to those mediating axonal retrograde signaling in neurons. This conclusion was supported not only by the cointernalization of NGF and TrkA, but also by the intracellular accumulation of TrkA autophosphorylated at its Shc binding site as seen in neurons (Ehlers et al., 1995; Bhattacharyya et al., 1997), and the preferential association of the internalized vesicles with an activated form of erk5 kinase, a critical neuronal retrograde signaling effector (Watson et al., 2001). The expression of PincherG68E completely and selectively blocked the cytoplasmic appearance of erk5 over erk1/2 kinases, although it had no effect on clathrin-mediated transferrin internalization, and allowed only a limited, low level of phospho-TrkA internalization. These results raise the possibility that the clathrin-independent internalization of NGF and TrkA by Pincher may selectively mediate the formation of the critical NGF/TrkA signaling endosome. The persistence of intracellular vesicles containing phospho-TrkA and phospho-erk5 kinase in Pincher-overexpressing PC12 cells, even after 24 h of NGF treatment, indicated that the internalized, activated NGF/TrkA complex was remarkably stable. This is a requisite characteristic for NGF/TrkA signaling endosomes in neurons, which must remain active during and after transport from the nerve terminal to the cell body. Pincher is expressed in peripheral neurons and brain, making Pincher an attractive candidate for mediating retrograde endosomal signaling in neurons.
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Materials and methods |
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TOGA analysis
TOGA was performed as described previously (Sutcliffe et al., 2000), herein using PC12 cytoplasmic polyA (+) RNA isolated from cells at various times after 1 min of NGF treatment.
Northern blot analysis
Total cellular RNA was isolated from cells, and Northern blot analysis was carried out as previously described (D'Arcangelo and Halegoua, 1993). Rat multitissue mRNA blots (CLONTECH Laboratories, Inc.) and total RNA blots from rat dorsal root ganglia, lung, and heart tissues were probed with UTP[-32P]-labeled antisense RNA probes using pHAL18, encoding 700 bp of 3' UTR region of Pincher mRNA and PIB15, encoding cyclophilin. HAL18/Pincher mRNA bands were analyzed using a PhophorImager and ImageQuant software (Molecular Dynamics). All values were normalized to the level of cyclophilin mRNA.
cDNA library construction and screening
PC12 Poly(A)+ mRNA isolated from PC12 cells 5 h after NGF treatment was also used to construct both random primed and oligo (dT)-primed ZAPII cDNA libraries (Stratagene). A pooled library consisted of 1.0 x 106 independent clones prior to amplification. Screening of 106 recombinants with a 32P-labeled HAL18 cDNA probe (oligo labeling kit; Amersham Pharmacia Biotech) resulted in the isolation of 15 positive clones. All 15 clones were sequenced and the overlapping sequences revealed a single full-length coding region for Pincher.
Recombinant cDNA constructs, expression and isolation of recombinant proteins, and generation of antibodies
A mammalian expression vector encoding HA-tagged Pincher was generated by subcloning full-length Pincher cDNA into the pCGN-HA vector between Xba1 and Kpn1 restriction sites. PincherG68E was generated by PCR-based mutagenesis, using primers designed to contain the mutation site and allow insertion (between Xba1 and BamH1 sites within Pincher) to generate pCGN-HA-PincherG68E. A bacterial expression vector encoding GST-Pincher was generated by subcloning full-length Pincher cDNA into pGEX-3x vector at the EcoR1 and BamH1 sites. GST-Pincher was purified from XL1-Blue (Stratagene) cells using glutathione-agarose beads (Sigma-Aldrich). Rabbit antiGST-Pincher polyclonal antibody was generated by Research Genetics, Inc. Myc-NGF was prepared from media of COS cells transfected with a myc-tagged NGF construct (Moller et al., 1998), 72 h after transfection and concentrated tenfold using ultra-free-15 Biomax-10k (Millipore) at 4°C. Cells were treated with myc-NGF at a concentration equivalent to 100 ng/ml NGF, as determined by bioassay. Cells were transfected using Lipofectamine 2000 (GIBCO BRL). 24 h after transfection, cells were plated on coverslips coated with 25µg/ml poly-L-lysine (Sigma-Aldrich) and 10 µg/ml laminin (BD Sciences Inc.). 5 h later, the medium was replaced with DME containing 1% horse serum and the cells cultured overnight.
Immunoprecipitation and Western blot analysis
Cells were washed in PBS and then lysed with lysis buffer (20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 50 mM NaF, 1 mM EDTA, 1 mM Na3VO4, 1 mM PMSF, 1 µg/ml leupeptin, 5 mM benzamidine, 1% NP-40). The lysates were clarified by centrifugation, and Pincher was immunoprecipitated from 500 µg of cell lysate for 4 h at 4°C using 5 µg of anti-Pincher antibody. The immunoprecipitates were subjected to SDS-10% PAGE and analyzed by Western blotting. Blots were probed with anti-Pincher (1:5,000) or anti-HA (1:200) antibodies and subsequently probed with secondary donkey antirabbit or donkey antimouse IgG antibodies conjugated with horseradish peroxidase (Amersham Pharmacia Bioteh) followed by ECL detection.
Immunofluorescence staining and imaging
After appropriate treatments as indicated in the text, cells were fixed and then subjected to double immunofluorescence staining. Cells were fixed in PBS containing 3.7% formaldehyde and 0.12 M sucrose for 10 min at room temperature for double staining using monoclonal anti-HA (1 µg/ul; Santa Cruz Biotechnology, Inc.) together with either anti-TrkA (1:250; Hempstead et al., 1992), or anti-phospho-erk5 (1:500; BioSource), or antiphosho-erk1/2 (1:250; New England Biolabs); or anti-Pincher (1:2,000) with monoclonal anti-myc (1 µg/ul, Sigma-Aldrich). Fixation in 4% paraformaldehyde was used for staining of anti-HA with antiP-Y490 (1:100; Bhattacharyya et al., 1997). Acetone fixation was used for anti-Pincher antibody with anti-TrkA mAb (1:100, MCTrkA; Santa Cruz Biotechnology, Inc.). Methanol fixation was used for anti-TrkA mAb with antiphospho-erk5. After fixation, cells were rinsed with PBS, blocked and permeabilized in Blotto (PBS containing 5% nonfat milk, 0.1% Triton) for anti-HA with anti-TrkA or antiP-Y490TrkA (also containing 1 mM vanadate), and for anti-Pincher with monoclonal anti-myc, and then incubated with primary antibody at 4°C overnight. Staining for monoclonal anti-TrkA, antiphospho-erk5 and antiphospho-erk1/2 was done according to New England Biolabs protocol, except that all solutions contained 1 mM vanadate. A collection of conjugated secondary antibodies were used including goat antimouse Alexa 488 or Alexa 546 (Molecular Probes), donkey antirabbit-Cy5 (Jackson) or goat antirabbit-Alexa 488. To visualize dextran internalization, cells were treated with Alexa 48810 kddextran (Molecular Probes) in DME at 1.25 mg/ml. For transferrin internalization, cells were treated with Alexa 633conjugated transferrin (25 µg/ml; Molecular Probes) in DME. Confocal images were obtained using a Zeiss LSM 510 laser scanning confocal microscope. Images were processed using Photoshop 6.0 software.
Electron microscopy and immunogold labeling
Fixation, dehydration, and embedding.
At 24 h posttransfection, TrkA-PC12 cells were further grown on ACLAR (Ted Pella) for 24 h. After NGF treatment, the cells were immersion fixed for 15 min in a cold mixture of 0.1 M phosphate buffer (PB, pH 7.4) containing 2% paraformaldehyde and 2% gluteraldehyde. Cells were rinsed 3 x 10 min in PB, osmicated (2% OsO4 for 1 h), rinsed 2 x 10 min in PB, rinsed 2 x 10 min in dH2O, en bloc stained with aqueous 1% uranylacetate for 1 h, rinsed 2 x 10 min in dH2O, dehydrated through an ascending series of ethanols, and embedded in Durcupan (Fluka) between sheets of ACLAR.
Postembedding immunogold labeling of Pincher.
Serial ultrathin sections (6090 nm) were cut on a Reichert Ultracut E ultratome and picked up on formvar-coated nickel slot grids. Postembedding immunogold labeling was done using a modification of the protocol of Phend et al. (1995). Sections were rinsed for 2 min in 0.1 M TRIS (pH 7.6) and 0.005% Tergitol NP-10 (Sigma-Aldrich), hereafter referred to as TRIS/T), followed by 5-s immersion in saturated (10%) sodium metaperiodate. Sections were rinsed 3 x 10 min in TRIS/T, incubated for 1 min at room temperature in 1% sodium borohydride, and rinsed 3 x 5 min in TRIS/T, and treated overnight with anti-Pincher antibody (TRIS/T, 1:30,000). Sections were rinsed 2 x 5 min and 1 x 30 min in TRIS/T, rinsed 5 min in TRIS/T at pH 8.2, and incubated for 1 h in goat antirabbit IgG-conjugated to 15 nm gold (Amersham Pharmacia Biotech), 1:25, TRIS/T, pH 8.2. Sections were rinsed 2 x 5 min in TRIS/T, rinsed 2 x 5 min dH2O, stained with 1% methanolic uranylacetate (10 min) and 0.3% aqueous lead citrate (5 min), and examined with a JEOL 1200 EX electron microscope (JEOL). In all control runs where either the primary or secondary antibody incubation step was skipped, all tissue specific anti-Pincher, immunogold labeling was eliminated.
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Footnotes |
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* Abbreviations used in this paper: GST, glutathione-S-transferase; HA, hemagglutinin; MAP, mitogen-activation protein; NGF, nerve growth factor; TOGA, total gene analysis; VAB, vesicle accumulating body.
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Acknowledgments |
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This work was supported by grants from the National Institutes of Health (NS18218 to S. Halegoua, and HL24103 to J.B. Cabot).
Submitted: 15 January 2002
Revised: 22 March 2002
Accepted: 1 April 2002
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References |
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