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Cell Growth & Differentiation Vol. 10, 805-812, December 1999
© 1999 American Association for Cancer Research

A TrkB/Insulin Receptor-related Receptor Chimeric Receptor Induces PC12 Cell Differentiation and Exhibits Prolonged Activation of Mitogen-activated Protein Kinase1

Karen S. Kelly-Spratt, Laura J. Klesse, Jussi Merenmies and Luis F. Parada2

Center For Developmental Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9133


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Insulin receptor-related receptor (IRR), an orphan receptor in the insulin receptor (IR) family of receptor tyrosine kinases, is primarily localized to neural crest-derived sensory neurons during embryonic development. Expression of IRR closely resembles that of the nerve growth factor receptor, TrkA. To analyze the signaling properties and function of IRR in PC12 cells, a TrkB/IRR hybrid receptor was used. In contrast to IR activation, brain-derived neurotrophic growth factor-mediated activation of the TrkB/IRR receptor resulted in differentiation rather than proliferation. Analysis of cytoplasmic substrates activated by the TrkB/IRR receptor indicates a signaling pathway similar to that of the IR. Mutagenesis studies further show that only TrkB/IRR receptors able to phosphorylate mitogen-activated protein kinase elicit a differentiation response. Our analysis indicates that prolonged kinetics of mitogen-activated protein kinase activation mediated by the TrkB/IRR chimeric receptor correlates with induction to differentiate.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
IRR3 was first identified from a low stringency screen of mammalian genomic libraries for novel members of the IR gene family of tyrosine kinases (1) . Like IR, IRR consists of an extracellular, cysteine-rich {alpha} chain that determines ligand specificity and a ß chain that spans the membrane and possesses intrinsic tyrosine kinase activity in its cytoplasmic region (2) . The two chains of IRR are synthesized as a single proreceptor molecule of Mr 174,000, which is then cleaved to yield the distinct {alpha} and ß chains of Mr 108,000 and Mr 66,000, respectively. The {alpha} and ß chains are tethered after cleavage by intramolecular disulfide bonds. Intermolecular disulfide bonds join the {alpha}ß-linked molecule into membrane-spanning dimers that constitute the active receptor (3) . Therefore, IRR is presumed to be an {alpha}2ß2 heterodimer complex, although this has not been formally demonstrated (4) .

The amino acid sequence of IRR exhibits high levels of identity with those of both IR and IGF-I receptor (1) . The cytoplasmic region of the ß subunit that defines the enzymatic domain for tyrosine-specific kinase activity exhibits the highest degree of identity, 79%. The regions between the kinase and transmembrane domains and the regions flanking the cysteine-rich domain are less similar, showing a range of 52–65% identity. However, there is a remarkable conservation of the cysteine residues, 88%. The most divergent region is the cytoplasmic COOH terminus, which has only 19–45% identity.

Studies to determine the ligand binding specificity of IRR have used chimeric receptors consisting of the extracellular domain of IRR on the backbone of IR. Although this chimeric receptor appeared to be expressed and processed normally, it did not bind labeled insulin (2) . In addition, unlabeled insulin and related proteins, IGF-I, IGF-II, and relaxin were incapable of stimulating the intrinsic tyrosine kinase activity of the chimeric receptor (2) . These results indicate that the ligand for IRR is unlikely to be either insulin or one of the known insulin-related molecules. Thus, IRR is an "orphan" receptor whose endogenous ligand remains unknown.

Previously, a chimeric receptor consisting of the extracellular domain of IR and the intracellular domain of IRR was described (2) . In CHO cells, the IR/IRR receptor stimulated biological responses similar to those of IR. Activation of the chimeric receptor mediated the stimulation of both the long-term and short-term response (i.e., thymidine incorporation and glucose uptake).

A number of IR substrates have been identified: (a) IRS-1, which in turn interacts with the SH2 domain of PI-3K (5) ; (b) a Mr 62,000 protein, which, when phosphorylated, is bound by GAP (ras GTPase activating protein; Ref. 6 ); and (c) SHC, which interacts with the SH2 domain of Grb-2 as well as the associated Ras-GTP exchanger, SOS (7) . In CHO cells, all three substrates were phosphorylated to a similar extent after insulin stimulation of the IR/IRR receptor and IR-induced phosphorylation (2) . This study indicates that IRR and IR have very similar transducing capabilities in CHO cells.

The full-length IRR has been cloned from the rat, guinea pig, and human (1 , 8) . Northern blot or reverse transcription-PCR studies show the highest IRR levels in rat stomach, kidney, muscle, and liver (2) . In situ hybridization revealed abundant IRR mRNA expression in neural crest-derived sensory and autonomic nerve ganglia as well as in the kidney during rat embryonic development (9 , 10) . This expression pattern is in contrast to a more widespread distribution of IR and IGF-I receptor mRNAs with the most abundant signals localized in the brain and the liver and muscle, respectively (9) . In neural crest derivatives, IRR gene expression is tightly coupled to that of the high-affinity NGF receptor, TrkA (4) . In addition, the IRR and TrkA coding regions are physically linked at the genome level and may share regulatory elements.4 The narrow tissue-specific coexpression of IRR and TrkA seen in the developing and mature nervous system prompted us to examine the functional signaling properties of IRR in neural cells.

PC12 (a rat pheochromocytoma tumor cell line) cells differentiate and resemble sympathetic neurons in response to NGF exposure (11) . PC12 cells express endogenous TrkA receptors, but not the related TrkB receptor. TrkB stably expressed in PC12 cells shows the same differentiation response: extension of neurites on addition of its ligands, BDNF and NT4/5 (12) . To analyze the potential function and signaling pathway of IRR in these cells, a hybrid receptor encoding cDNA with the extracellular domain of TrkB and the transmembrane and intracellular domains of IRR was constructed and expressed in PC12 cells. The biological response to the BDNF-stimulated chimeric receptor was assayed in the resulting clonal cell lines. The results were compared directly to the endogenous TrkA and IRs as well as PC12 cells expressing either ectopic TrkB or IR. In addition, specific tyrosine residues in the IRR kinase and juxtamembrane domains were assessed for a role in activation of the receptor and regulation of downstream signaling.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
To analyze the intracellular cascade used by IRR in the absence of a known ligand, a chimeric receptor containing the extracellular domain of TrkB and the transmembrane and intracellular domains of IRR was constructed (Fig. 1Citation ; see "Materials and Methods") and transfected into PC12 cells. G418-resistant colonies were isolated and screened for expression of the TrkB/IRR protein. Expression of the TrkB/IRR chimeric receptor was detected in each clonal cell line by WGA-Sepharose precipitation and Western transfer analysis. A Mr 110,000 protein band was detected using either an IRR COOH-terminal polyclonal antibody or a TrkB NH2-terminal polyclonal antibody (data not shown). Fig. 1Citation demonstrates that the chimeric TrkB/IRR receptor is phosphorylated on tyrosine residues in response to BDNF. Untransfected PC12 cells treated in parallel showed no response, whereas PC12 cells expressing exogenous TrkB also became phosphorylated (12) .



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Fig. 1. TrkB/IRR chimeric receptor construction and expression in PC12 cells. The TrkB extracellular domain was fused to the transmembrane and intracellular domains of IRR to create a chimeric expression plasmid. Stable PC12 cells were generated and analyzed for protein expression by Western blot analysis. Cells were stimulated with 50 ng/ml BDNF for 5 min, lysates were collected, and protein was immunoprecipitated with a phosphotyrosine-specific antibody and observed with a TrkB NH2-terminal-specific antibody.

 
Multiple clonal cell lines were established expressing various levels of TrkB/IRR, ranging from high expression to levels comparable to endogenous TrkA or IR (x104 receptors/cell; Refs. 13 and 14 ). Subsequent experiments were performed using the TrkB/IRR cell line that exhibited receptor levels similar to those of a TrkB-expressing PC12 cell line (12) as well as PC12/TrkA and PC12/IR control cell lines.

A dose-response experiment was performed to determine the activity of BDNF on the TrkB/IRR receptor. Cells were incubated for 5 min with neurotrophin concentrations ranging from 10–200 ng/ml and subjected to Western analysis with an antiphophotyrosine antibody. TrkB/IRR receptor phosphorylation is induced maximally by BDNF at 50 ng/ml, and half-maximal response was obtained with concentrations of 5–10 ng/ml (Fig. 2a)Citation . This level of BDNF required for receptor activation is similar with that observed for TrkB stimulation in PC12/TrkB cells (Fig. 2a)Citation .



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Fig. 2. Dose-response and time course of BDNF stimulation of TrkB/IRR. a, PC12 TrkB/IRR-expressing cells and PC12 TrkB-expressing cells were incubated for 5 min with quantities of BDNF ranging from 10–200 ng/ml. PC12IR cells were incubated for 5 min with quantities of insulin ranging from 100 ng/ml to 5 mg/ml. Activated receptor is observed with an antiphosphotyrosine antibody. b, PC12 TrkB/IRR-expressing cells were incubated for 1 min to 1 h with 50 ng/ml BDNF. Activated receptor is observed with the antiphosphotyrosine antibody.

 
Next, a time course experiment was performed to determine the timing of BDNF exposure required for maximal levels of TrkB/IRR receptor phosphorylation. Cells were incubated for 1 min to 1 h with 50 ng/ml BDNF. Tyrosine phosphorylation of TrkB/IRR was detected within 1 min of BDNF addition, and maximal levels of phosphorylation were reached within 5–10 min (Fig. 2b)Citation . These results were similar to those reported previously for endogenous TrkA (15) and PC12/TrkB cells (12) .

BDNF Stimulation Induces Differentiation.
Previous IR studies in PC12 cells have demonstrated that receptor activation leads to enhanced cellular proliferation (16) . In contrast, NGF activation of TrkA results in differentiation and neurite extension (15) . To examine the biological response of TrkB/IRR in PC12 cells, BDNF (50 ng/ml) was added to either serum-containing or serum-free cultured cells that were observed for 7–10 days. Differentiation was observed by 3 days in the PC12 TrkB/IRR cells, with an extensive neurite network achieved by 10 days under both culture conditions (Fig. 3)Citation . Untransfected PC12 cells, PC12 TrkB/IRR, PC12 TrkA, and PC12 IR stimulated with NGF (50 ng/ml) all showed TrkA-mediated neurite outgrowth in the same culture conditions. Untransfected PC12 cells and PC12 IR cells stimulated with insulin (1 µg/ml) showed short dendrite-like extensions only in the absence of serum but failed to develop the neurite network seen with neurotrophin stimulation (Fig. 3)Citation .



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Fig. 3. Biological response of TrkB/IRR to BDNF in PC12 cells. PC12, PC12IR, and PC12TrkB/IRR cells were stimulated with 50 ng/ml BDNF under both serum and serum-free growth conditions. Cells were observed over a 10-day period for differentiation or increased cellular proliferation. Cells were stimulated with 50 ng/ml NGF for differentiation control or with 1 µg/ml insulin for a proliferation control.

 
To determine the dose of BDNF required for differentiation, PC12 TrkB/IRR cells were incubated at concentrations ranging from 10–200 ng/ml and then observed for 10 days. A dose of 50 ng/ml gave maximal response, with the production of an extensive neurite network, whereas half-maximal response was seen at 10 ng/ml (Table 1)Citation . This biological response correlates with the previous receptor phosphorylation data and indicates that, physiologically, functional IRR activation more closely resembles Trk receptors than IR, despite its homology to the latter.


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Table 1 Percentage of neurite outgrowth of PC12, PC12TrkB, and PC12 TrkB/IRR

 
Tyrosine Residues Necessary for TrkB/IRR Activation.
We next examined downstream substrates of the activated TrkB/IRR receptor. Cells were lysed after the addition of BDNF and subjected to immunoprecipitation and Western analysis with various antibodies specific to proteins identified previously as substrates of either activated TrkA or IR. IRS-1 and IRS-2 are specifically phosphorylated in response to IR activation (5 , 17) , whereas SNT and PLC-{gamma} are known substrates of TrkA (18, 19, 20, 21) . As summarized in Fig. 4aCitation , the TrkB/IRR profile of cytoplasmic substrates was indistinguishable from that of substrates of IR. As an example, Fig. 4bCitation illustrates the analysis for PLC-{gamma}.



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Fig. 4. Comparison of TrkB/IRR signaling substrates to the IR and TrkA receptor in PC12 cells. Immunoprecipitation with antibodies specific to various cytoplasmic proteins shown previously to be involved in downstream signaling were used on PC12 and PC12 TrkB/IRR cell lysates. Western blots were probed with a phosphotyrosine-specific antibody. a, table shows the results of Western blot analysis. p, phosphorylated on tyrosine residues. b, Western blot analysis sample showing PLC-{gamma} phosphorylation.

 
To determine the intracellular tyrosine residues important for differentiation mediated by activation of TrkB/IRR in PC12 cells, the tyrosine residues in the juxtamembrane, kinase, and COOH terminus domains of the TrkB/IRR expression plasmid were replaced with phenylalanine residues by site-directed mutagenesis (see "Materials and Methods" for details). The replaced tyrosine residues correspond to amino acids 934, 1079, 1114, 1118, 1119, 1114/1118, 1114/1119, 1118/1119, 1114/1118/1119, 1167, 1210, 1211, and 1210/1211. In addition, the ATP binding site was mutated (Fig. 5a)Citation . The resulting mutant plasmids were used to derive stable transfected PC12 cell lines, and each cell line was examined by Northern and Western blot analysis for mRNA fidelity and receptor protein expression. All cell lines had transcripts of the predicted size (3.0 kb) and expressed a receptor of Mr 110,000 (data not shown).



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Fig. 5. a and b, assessment of mutant TrkB/IRR activation and phosphorylation of downstream substrates. Tyrosine residues in the juxtamembrane, kinase, and COOH-terminal domains were mutated to phenylalanine residues, and stable PC12 cell lines were made. Analysis of the effects of the mutations was performed using immunoprecipitation and Western blot analysis of each mutant receptor compared to the wild-type TrkB/IRR-expressing PC12 cell line. Tyrosine phosphorylation of each receptor, IRS-1, and MAPK was assessed. Biological response was assayed by BDNF-stimulated neurite outgrowth. a, tabulation of Western blot analysis and the neurite outgrowth assay (+, phosphorylation; -, no phosphorylation). b, Western blots showing phosphorylation of receptor, IRS-1, or MAPK for each mutant TrkB/IRR cell line.

 
Each mutant TrkB/IRR cell line was stimulated with BDNF (50 ng/ml) for 5 min, and cell lysates were subjected to antiphosphotyrosine analysis. The results indicate that single tyrosine mutations do not alter the ability of the receptor to become phosphorylated. However, each of the double and triple mutations in the kinase domain abolished receptor phosphorylation, and alteration of the ATP binding site resulted in loss of receptor phosphorylation (Fig. 5b)Citation .

To examine the ability of the various mutant TrkB/IRR receptors to signal, the phosphorylation state of two downstream substrates, IRS-1 and MAPK, was assessed. Each mutant cell line was stimulated for 5 min with BDNF (50 ng/ml), and lysates were collected, immunoprecipitated with an IRS-1 COOH-terminal polyclonal antibody, and subjected to Western blot analysis with an antiphosphotyrosine antibody. The results show that those mutations that inhibited receptor phosphorylation also showed no IRS-1 phosphorylation (amino acids 1114/1118, 1114/1119, 1118/1119, 1114/1118/1119, and the ATP site; Fig. 5bCitation ). A phosphospecific MAPK polyclonal antibody was used to examine activation. Mutations that adversely affected receptor phosphorylation also abolished MAPK phosphorylation. In addition, tyrosine 934, which, on the basis of IR homology, corresponds to the IRS-1 binding site, was negative for both IRS-1 activation and MAPK phosphorylation (Fig. 5b)Citation . The two tyrosines located at the COOH terminus, residues 1210 and 1211, exhibited complex results. When mutated singly, 1210 abrogated IRS-1 phosphorylation, whereas 1211 retained phosphorylation. However, each of the single mutants was also defective for MAPK phosphorylation, whereas the double mutant (1210/1211) retained MAPK activity (Fig. 5b)Citation .

A functional neurite outgrowth assay was performed with each PC12 TrkB/IRR mutant cell line in response to BDNF. Only the cell lines with receptors mutated at residues 1079, 1167, and 1210/1211 were able to elicit a differentiation response, indicating a correlation between the requirement for MAPK activity and differentiation (see Fig. 5aCitation ).

Functional Downstream Substrates.
Based on the preceding results, we next examined functional downstream effectors of the activated chimeric receptor. Recombinant adenoviruses encoding DN versions of Ras, Raf, mitogen-activated protein/ERK kinase, and ERK proteins (22) were introduced into PC12 TrkB/IRR cells. We have previously demonstrated the ability of these recombinant adenoviruses to inhibit endogenous ERK phosphorylation and NGF-TrkA receptor-mediated neurite outgrowth in PC12 cells (22) . To control against adenoviral gene expression-mediated effects, a recombinant adenovirus harboring an inactive form of Raf (kinase mutant) was used. As indicated in Fig. 6Citation , each of the DN adenoviruses blocked neurite outgrowth in a dose-dependent manner. When tested in serum-free culture conditions, the DN adenoviruses did not appear to inhibit survival of PC12 TrkB/IRR cells. These data indicate that the Ras-ERK pathway is required for IRR-mediated PC12 cell differentiation but not for survival.



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Fig. 6. Adenovirus-expressing DN Ras-MAPK pathway substrates inhibit differentiation of BDNF-stimulated PC12 TrkB/IRR cells. Stable TrkB/IRR-expressing PC12 cells were infected with DN Ras, Raf, mitogen-activated protein/ERK kinase, and ERK2 adenovirus at the MOI indicated for 48 h. Cells were stimulated with 50 ng/ml BDNF and observed for neurite outgrowth over 10 days. {square}, 25 MOI; , 50 MOI, , 100 MOI; {blacksquare}, 200 MOI.

 
Kinetics of MAPK Activation.
Previous studies in PC12 cells have revealed a difference in the kinetics of MAPK activation when the cells are subjected to insulin, EGF, or NGF. Whereas insulin or EGF induced a transient MAPK activation, NGF generated a sustained activation of this downstream substrate (16) . Thus, the differential kinetics of MAPK activation may underlie the type of response (proliferation versus differentiation) in these cells. To investigate whether the differentiation response mediated by an activated TrkB/IRR is a result of the duration of MAPK phosphorylation, a time course experiment was performed. PC12 TrkB/IRR and PC12 TrkB cells were incubated for 5 min to 1 h with BDNF (50 ng/ml). PC12 IR cells were incubated for the same time period with insulin (1 µg/ml). Phosphorylation of MAPK was detected within 1 min of ligand addition in the three cell lines, with maximal levels of phosphorylation reached within 5 min (Fig. 7)Citation . Maximal phosphorylation of MAPK was sustained for 1 h in both the TrkB/IRR- and TrkB-expressing PC12 cells, whereas it declined rapidly in PC12 IR cells (Fig. 7)Citation . Therefore, TrkB/IRR-induced differentiation may be the result of prolonged versus transient MAPK phosphorylation.



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Fig. 7. Time course of MAPK phosphorylation. PC12TrkB/IRR, PC12TrkB, and PC12IR were stimulated with either 50 ng/ml BDNF or 1 µg/ml insulin and analyzed for MAPK phosphorylation from 5 min to 1 h. Analysis with anti-ERK1 shows equivalent protein levels for all time points.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
In this study, we investigated the signaling and potential function of the orphan IRR receptor in a model neuronal system. A hybrid TrkB/IRR receptor was constructed, expressed in PC12 cells, and demonstrated to respond specifically to BDNF. In contrast to insulin-mediated activation of IR, BDNF activation of TrkB/IRR in PC12 cells initiates a differentiation response similar to that of TrkA stimulated by NGF.

IR and IRR show an overall 80% structural and sequence identity that is greatest in the kinase domain (1) . Comparison of the TrkA and IR signaling pathways show that both are capable of phosphorylation and activation of the Ras-MAPK signaling pathway (23 , 24) . The major difference identified in the signaling substrate cascades of TrkA and IR is the use of different adaptor molecules directly downstream of the activated receptors, IRS-1 for IR and SHC for TrkA. Among the substrates we examined, IRR was identical to IR in its signaling capacities and distinct from Trk receptor signaling. Paradoxically, TrkB/IRR functionally mimics TrkA or TrkB signaling in PC12 cells. SNT has been identified as a specific target of factor-induced differentiation (18) . The TrkB/IRR chimeric receptor does not phosphorylate SNT in the process of mediating PC12 differentiation, indicating that SNT is not responsible for this functional difference.

Another approach used to understanding the phenotypic differences between activated IR and IRR in PC12 cells was to examine the contribution of specific tyrosine residues in the TrkB/IRR chimera proposed to be involved in activation and downstream signaling. By site-directed mutagenesis, we show that the tyrosine residue at position 934 appears to be important for phosphorylation of the adaptor IRS-1. This is consistent with results obtained from studies of IR mutated at the corresponding tyrosine residue (25, 26, 27) .

The COOH-terminal double mutation (1210/1211) of IRR does not affect either receptor phosphorylation or downstream signaling, whereas mutation of either moiety alone leads to receptor inactivation. In IR, these residues have been considered as negative regulators of signaling because on dual mutation, enhanced MAPK activity was seen in rat fibroblasts (28) . No studies have been reported on the mutation of each IR terminal tyrosine alone. In our study, examination of each single COOH-terminal tyrosine of IRR suggests each tyrosine may act independently. Mutation of tyrosine 1210 resulted in loss of IRS-1 phosphorylation. IRS-1 phosphorylation occurs with the absence of tyrosine 1211, but no MAPK activity was seen. Thus each tyrosine mutated alone inactivates aspects of receptor signaling and function in PC12 cells, but inactivation of both sites leaves receptor signaling and function intact. The TrkA receptor contains corresponding COOH-terminal tyrosine residues that are binding sites for two distinct molecules, PLC-{gamma} and PI-3K (20 , 21) . However, PLC-{gamma} is not phosphorylated by activated IRR, suggesting that a different protein may bind and become activated to promote the differentiation response.

It has been postulated that the difference between proliferation and differentiation of PC12 cells is the intensity of the activation signal or the duration of the signal with translocation to the nucleus. In particular, MAPK phosphorylation by NGF stimulation of TrkA is sustained and leads to nuclear translocation, whereas activation by insulin or EGF is transient and does not lead to nuclear translocation (16 , 29) . Differentiation has been reported only with high overexpression of either IR or EGFR (29 , 30) . However, TrkB/IRR elicits a differentiation response even when present at levels equivalent to endogenous EGFR, TrkA, or IR, suggesting that differentiation is not a phenomenon of overexpression. In addition, we have shown that stimulation of PC12 cells overexpressing the TrkB/IRR receptor results in a sustained MAPK phosphorylation similar to TrkB-expressing PC12 cells or endogenous TrkA-activated PC12 cells, in contrast to a transient MAPK phosphorylation seen with PC12 cells overexpressing IR.

We have shown that PC12 cells expressing equivalent levels of IR versus TrkB or TrkB/IRR have a different biological response when ligand stimulated. One explanation may be that only a fraction of IRs are occupied in the presence of excess ligand, although all receptors are potentially fully functional (31) . On ligand binding, insulin-IR complexes are internalized, sequestered from the plasma membrane, and concentrated in the endosome, where various functions including receptor sorting (recycling or degradation), ligand processing and targeting, and signal transduction and termination occur (32) . Internalization mediates IR activity down-regulation and attenuation of insulin sensitivity. IR maintains its phosphorylation state and tyrosine activity after dissociation of insulin from the {alpha} subunit, making it necessary for dephosphorylation of the ß subunit tyrosine residues to deactivate the intrinsic kinase activity before recycling (33) . The combination of fewer activated receptors and sequestration of phosphorylated receptor may play a crucial role in regulating IR activity and signal transduction. Analysis of IRR internalization may lead to an understanding of the regulation of IRR activation. Other results do not support the hypothesis that reduced internalization and hence prolonged activation of the TrkB/IRR hybrid receptor might account for its differences with IR. We have constructed a chimeric TrkB/IR receptor and directly compared its function to that of the TrkB/IRR receptor. These studies used adenovirus-mediated transduction into PC12 cells and primary sympathetic neurons. Whereas the TrkB/IRR receptor mediated survival of sympathetic neurons in the absence of neurotrophins, the similarly constructed TrkB/IR receptor had no such effect.5

The activity of TrkB/IRR is dependent on phosphorylation of specific tyrosine residues, similar to that of IR to elicit functional signaling. However, the functional outcome resembles TrkA activation, including prolonged phosphorylation of MAPK. Continued analysis of IRR versus TrkA signaling may shed some light on the key features of signal transduction that push the cell to proliferate versus differentiate.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Construction of the TrkB/IRR Chimeric Receptor.
The intracellular domain of IRR was isolated from a pBK-CMVIRR plasmid by MluI-KpnI double restriction enzyme digestion. The extracellular domain of TrkB was isolated from a pBluescriptTrkBFL plasmid by BamHI-NdeI double restriction enzyme digestion. The transmembrane domain of IRR was isolated and linked to the 3' end of the TrkB extracellular domain by a modified PCR protocol with "bridge" fragments from two different plasmids. The primer for the IRR transmembrane domain was 5'-CCATCGATGGCTCCTCCACTCCTATCTGATCTCTG-3', the TrkB extracellular domain was 5'-GGGCTCTAGATCTCCATTGGTCCGGGGGTTGATTTC-3', and the bridge fragment was 5'-CCCACTCATATGAATAATGGA-3'. PCR of the bridge fragment required the use of a Hot Start (Molecular Bio-Products, Inc.) wax seal for specific priming. The resulting PCR product was double restriction enzyme digested with NdeI-MluI and then ligated to the expression vector containing the 5' TrkB and the 3' IRR fragments.

Identification of TrkB/IRR-expressing Cell Lines.
The pMexneoTrkB/IRR plasmid was transfected into PC12 cells by electroporation using a Bio-Rad Gene Pulser. PC12 cells were grown in DMEM with 10% heat-inactivated horse serum (Hyclone) and 5% heat-inactivated fetal bovine serum (Life Technologies, Inc.), 1% penicillin/streptomycin (Life Technologies, Inc.), and 1% sodium pyruvate (Life Technologies, Inc.). G418-resistant colonies were selected and analyzed for expression of the TrkB/IRR receptor by WGA-Sepharose (Pharmacia) precipitation and Western blot transfer analysis. Cells were washed in PBS and then lysed with NP40 lysis buffer [150 mM NaCl, 50 mM Tris (pH 8), and 1% NP40] containing protease inhibitors (aprotinin, pepstatin, phenylmethylsulfonyl fluoride, and leupeptin). Receptors were captured on WGA-Sepharose beads, washed three times in lysis buffer, and run on an 8% SDS-polyacrylamide gel. Proteins were transferred to a polyvinylidene difluoride membrane by Western blot and analyzed with either a NH2-terminal-specific polyclonal TrkB antibody or a COOH-terminal-specific polyclonal IRR antibody.

Analysis of Phosphorylated TrkB/IRR and Downstream Substrates.
Activation of the BDNF-stimulated TrkB/IRR receptor was analyzed by immunoprecipitation of cell lysates with a monoclonal phosphotyrosine antibody (Upstate Biotechnology), capture to protein A-Sepharose beads (Sigma and Pharmacia), and SDS-PAGE. Western blot analysis involved either the IRR or TrkB antibodies. Tyrosine phosphorylation of downstream substrates in response to activation of the chimeric receptor involved immunoprecipitation of stimulated cell lysates with antibodies specific to IRS-1 (Upstate Biotechnology); SHC, NCK, SHP2, and IRS-2 (Santa Cruz Biotechnology); PI-3K and PLC-{gamma} (Transduction Laboratories); and SNT (yeast p13 suc1 agarose conjugate; Upstate Biotechnology), and then Western blot analysis was performed using the phosphotyrosine-specific monoclonal antibody. Phosphorylation of MAPK was analyzed using a phosphospecific MAPK antibody (Promega) and ERK1 (Santa Cruz Biotechnology) against whole cell lysates.

Neurite Outgrowth Assay.
PC12 cells stably expressing the transfected TrkB/IRR receptor were plated at a density of 1 x 104 cells/60-mm tissue culture dish. At 24 h after plating, the media were replaced with DMEM containing 0.05% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin. BDNF was added at a concentration of 50 ng/ml, and cells were incubated for up to 10 days. Cells were observed for either differentiation (ability to form neurite extensions greater than three cell body lengths) or increased proliferation, measured by increased cell count on 60-mm tissue culture dishes that have grids outlined on their bases. Control cells were stimulated with 50 ng/ml NGF for the differentiation response or 1 µg/ml insulin for the proliferation response.

Generation of Mutant TrkB/IRR Clonal Cell Lines.
The tyrosine residues in the juxtamembrane, kinase domain, and COOH terminus were changed to phenylalanine residues by site-directed mutagenesis using the Chameleon Site-directed Mutagenesis Kit (Stratagene). Tyrosine residues mutated are 934, 1079, 1114, 1118, 1119, 1114/1118, 1114/1119, 1118/1119, 1114/1118/1119, 1167, 1210, 1211, and 1210/1211. The ATP binding site was also mutated. The resulting plasmids were transfected into PC12 cells, and clonal cell lines were selected with 500 µg/ml G418. Each mutant cell line was examined by Northern and Western blot analysis for mRNA fidelity and receptor protein expression.

Assessment of Activation, Biological Response, and Downstream Signaling.
Each mutant cell line was stimulated with 50 ng/ml BDNF (Regeneron Pharmaceuticals) for 5 min, and cell lysates were subjected to immunoprecipitation and Western blot analysis using either an antiphosphotyrosine monoclonal antibody (Upstate Biotechnology), an IRS-1 COOH-terminal-specific polyclonal antibody (Upstate Biotechnology), or a phosphospecific MAPK polyclonal antibody (Promega). Lysates were incubated with 1 µg of antibody for 12–18 h at 4°C, and then proteins were bound to protein A-Sepharose beads (Pharmacia), washed three times with lysis buffer, and run on an 8% SDS-PAGE.

The biological response to BDNF stimulation of each mutant TrkB/IRR cell line was examined by use of the neurite outgrowth assay as described above. Each mutant cell line was stimulated with 50 ng/ml BDNF and observed over a 10-day period for the extension of neurites.


    Acknowledgments
 
We thank the members of the Parada laboratory for helpful discussions and Kim Meyers for assistance in preparing the manuscript.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was funded by NINDS R01-NS33199 and the Excellence in Education Endowment in Developmental Biology (to L. F. P.). Back

2 To whom requests for reprints should be addressed. Phone: (214) 648-1951; Fax: (214) 648-1960; E-mail: parada{at}utsw.swmed.edu Back

3 The abbreviations used are: IRR, insulin receptor-related receptor; IR, insulin receptor; DN, dominant negative; MAPK, mitogen-activated protein kinase; BDNF, brain-derived neurotrophic growth factor; NGF, nerve growth factor; IGF, insulin-like growth factor; CHO, Chinese hamster ovary; PI-3K, phosphatidylinositol 3'-kinase; WGA, wheat germ agglutinin; ERK, extracellular signal-regulated kinase; EGF, epidermal growth factor; MOI, multiplicity of infection; IRS-1, insulin receptor substrate-1; PLC-{gamma}, phospholipase C-{gamma}. Back

4 L. Ma, J. Merenmies, and L. F. Parada. Identification of the transcriptional regulators for neuronal expression of TrkA and IRR, manuscript in preparation. Back

5 K. S. Kelly-Spratt, L. J. Klesse, and L. F. Parada. A BDNF activated TrkB/IRR receptor chimera promotes survival of sympathetic neurons through ras and PI3 kinase signalling, manuscript in preparation. Back

Received for publication 7/27/99. Revision received 11/ 4/99. Accepted for publication 11/ 4/99.


    References
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 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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