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• W2010423785 abstract "A key issue regarding the role of α6β4 in cancer biology is the mechanism by which this integrin exerts its profound effects on intracellular signaling, including growth factor-mediated signaling. One approach is to evaluate the intrinsic signaling capacity of the unique β4 intracellular domain in the absence of contributions from the α6 subunit and tetraspanins and to assess the ability of growth factor receptor signaling to cooperate with this domain. Here, we generated a chimeric receptor composed of the TrkB extracellular domain and the β4 transmembrane and intracellular domains. Expression of this chimeric receptor in β4-null cancer cells enabled us to assess the signaling potential of the β4 intracellular domain alone or in response to dimerization using brain-derived neurotrophic factor, the ligand for TrkB. Dimerization of the β4 intracellular domain results in the binding and activation of the tyrosine phosphatase SHP-2 and the activation of Src, events that also occur upon ligation of intact α6β4. In contrast to α6β4 signaling, however, dimerization of the chimeric receptor does not activate either Akt or Erk1/2. Growth factor stimulation induces tyrosine phosphorylation of the chimeric receptor but does not enhance its binding to SHP-2. The chimeric receptor is unable to amplify growth factor-mediated activation of Akt and Erk1/2, and growth factor-stimulated migration. Collectively, these data indicate that the β4 intracellular domain has some intrinsic signaling potential, but it cannot mimic the full signaling capacity of α6β4. These data also question the putative role of the β4 intracellular domain as an “adaptor” for growth factor receptor signaling. A key issue regarding the role of α6β4 in cancer biology is the mechanism by which this integrin exerts its profound effects on intracellular signaling, including growth factor-mediated signaling. One approach is to evaluate the intrinsic signaling capacity of the unique β4 intracellular domain in the absence of contributions from the α6 subunit and tetraspanins and to assess the ability of growth factor receptor signaling to cooperate with this domain. Here, we generated a chimeric receptor composed of the TrkB extracellular domain and the β4 transmembrane and intracellular domains. Expression of this chimeric receptor in β4-null cancer cells enabled us to assess the signaling potential of the β4 intracellular domain alone or in response to dimerization using brain-derived neurotrophic factor, the ligand for TrkB. Dimerization of the β4 intracellular domain results in the binding and activation of the tyrosine phosphatase SHP-2 and the activation of Src, events that also occur upon ligation of intact α6β4. In contrast to α6β4 signaling, however, dimerization of the chimeric receptor does not activate either Akt or Erk1/2. Growth factor stimulation induces tyrosine phosphorylation of the chimeric receptor but does not enhance its binding to SHP-2. The chimeric receptor is unable to amplify growth factor-mediated activation of Akt and Erk1/2, and growth factor-stimulated migration. Collectively, these data indicate that the β4 intracellular domain has some intrinsic signaling potential, but it cannot mimic the full signaling capacity of α6β4. These data also question the putative role of the β4 intracellular domain as an “adaptor” for growth factor receptor signaling. The α6β4 integrin is a structural and functional anomaly among the integrin family of receptors. This integrin, which is expressed primarily on the basal surface of epithelia and in a few other cell types, is defined as an adhesion receptor for most of the known laminins (1Kikkawa Y. Sanzen N. Fujiwara H. Sonnenberg A. Sekiguchi K. J. Cell Sci. 2000; 113: 869-876Crossref PubMed Google Scholar, 2Mercurio A.M. Trends Cell Biol. 1995; 5: 419-423Abstract Full Text PDF PubMed Scopus (199) Google Scholar, 3Pouliot N. Connolly L.M. Moritz R.L. Simpson R.J. Burgess A.W. Exp. Cell Res. 2000; 261: 360-371Crossref PubMed Scopus (45) Google Scholar). The distinguishing structural feature of α6β4 is the atypical intracellular domain of the β4 subunit. Two pairs of fibronectin type III repeats separated by a connecting segment characterize this domain, and it is distinct both in size (∼1000 amino acids) and structure from any other integrin subunit (4Borradori L. Sonnenberg A. Curr. Opin. Cell Biol. 1996; 8: 647-656Crossref PubMed Scopus (197) Google Scholar). Although the α6β4 integrin provides a well characterized adhesive function in normal epithelial cells by anchoring the epithelium to its underlying basement membrane, the carcinoma-associated functions of this integrin are becoming increasingly recognized (5Lipscomb E.A. Mercurio A.M. Cancer Metastasis Rev. 2005; 24: 413-423Crossref PubMed Scopus (115) Google Scholar). Importantly, the expression of this integrin is often maintained as epithelial structures dissociate during the initiation and progression of carcinomas, and, consequently, many carcinomas express α6β4 (6Mercurio A.M. Rabinovitz I. Semin. Cancer Biol. 2001; 11: 129-141Crossref PubMed Scopus (195) Google Scholar, 7Rabinovitz I. Mercurio A.M. Biochem. Cell Biol. 1996; 74: 811-821Crossref PubMed Scopus (102) Google Scholar). Numerous studies by our groups and others have revealed that α6β4 can facilitate the ability of carcinoma cells to migrate, invade, and resist apoptotic stimuli (8Bachelder R.E. Ribick M.J. Marchetti A. Falcioni R. Soddu S. Davis K.R. Mercurio A.M. J. Cell Biol. 1999; 147: 1063-1072Crossref PubMed Scopus (157) Google Scholar, 9Chao C. Lotz M.M. Clarke A.C. Mercurio A.M. Cancer Res. 1996; 56: 4811-4819PubMed Google Scholar, 10Chung J. Bachelder R.E. Lipscomb E.A. Shaw L.M. Mercurio A.M. J. Cell Biol. 2002; 158: 165-174Crossref PubMed Scopus (170) Google Scholar, 11Gambaletta D. Marchetti A. Benedetti L. Mercurio A.M. Sacchi A. Falcioni R. J. Biol. Chem. 2000; 275: 10604-10610Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 12Jauliac S. Lopez-Rodriguez C. Shaw L.M. Brown L.F. Rao A. Toker A. Nat. Cell Biol. 2002; 4: 540-544Crossref PubMed Scopus (347) Google Scholar, 13Owens D.M. Romero M.R. Gardner C. Watt F.M. J. Cell Sci. 2003; 116: 3783-3791Crossref PubMed Scopus (75) Google Scholar, 14Rabinovitz I. Toker A. Mercurio A.M. J. Cell Biol. 1999; 146: 1147-1160Crossref PubMed Scopus (187) Google Scholar, 15Weaver V.M. Lelievre S. Lakins J.N. Chrenek M.A. Jones J.C. Giancotti F. Werb Z. Bissell M.J. Cancer Cell. 2002; 2: 205-216Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar, 16Zahir N. Lakins J.N. Russell A. Ming W. Chatterjee C. Rozenberg G.I. Marinkovich M.P. Weaver V.M. J. Cell Biol. 2003; 163: 1397-1407Crossref PubMed Scopus (151) Google Scholar). More recently, α6β4 has been implicated in the genesis of squamous and breast carcinomas (17Dajee M. Lazarov M. Zhang J.Y. Cai T. Green C.L. Russell A.J. Marinkovich M.P. Tao S. Lin Q. Kubo Y. Khavari P.A. Nature. 2003; 421: 639-643Crossref PubMed Scopus (476) Google Scholar, 18Guo W. Pylayeva Y. Pepe A. Yoshioka T. Muller W.J. Inghirami G. Giancotti F.G. Cell. 2006; 126: 489-502Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar, 19Lipscomb E.A. Simpson K.J. Lyle S.R. Ring J.E. Dugan A.S. Mercurio A.M. Cancer Res. 2005; 65: 10970-10976Crossref PubMed Scopus (77) Google Scholar). The ability of α6β4 to impact these diverse functions results largely from its effects on multiple signaling pathways, including phosphatidylinositol 3-kinase/Akt and MAPK, 2The abbreviations used are: MAPK, mitogen-activated protein kinase; BDNF, brain-derived neurotrophic factor; DMEM, Dulbecco's modified Eagle's medium; shRNA, short hairpin RNA; Ab, antibody; mAb, monoclonal antibody; Erk, extracellular signal-regulated kinase; ALLN, N-acetyl-Leu-Leu-norleucinal; PBS, phosphate-buffered saline; BSA, bovine serum albumin; p75NTR, p75 neurotrophic receptor; SFK, Src family kinase; HGF, hepatocyte growth factor. 2The abbreviations used are: MAPK, mitogen-activated protein kinase; BDNF, brain-derived neurotrophic factor; DMEM, Dulbecco's modified Eagle's medium; shRNA, short hairpin RNA; Ab, antibody; mAb, monoclonal antibody; Erk, extracellular signal-regulated kinase; ALLN, N-acetyl-Leu-Leu-norleucinal; PBS, phosphate-buffered saline; BSA, bovine serum albumin; p75NTR, p75 neurotrophic receptor; SFK, Src family kinase; HGF, hepatocyte growth factor. a process that may result from its association with specific growth factor receptors, tetraspanins, and possibly other molecules (5Lipscomb E.A. Mercurio A.M. Cancer Metastasis Rev. 2005; 24: 413-423Crossref PubMed Scopus (115) Google Scholar). The dichotomy of α6β4 function is summarized best by the hypothesis that α6β4 switches from a mechanical adhesive device into a signaling competent receptor during the progression from normal epithelium to invasive carcinoma (5Lipscomb E.A. Mercurio A.M. Cancer Metastasis Rev. 2005; 24: 413-423Crossref PubMed Scopus (115) Google Scholar, 20Santoro M.M. Gaudino G. Marchisio P.C. Dev. Cell. 2003; 5: 257-271Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). A key issue regarding the role of α6β4 in carcinoma biology is the mechanism by which this integrin exerts its profound effects on intracellular signaling. Given that α6β1 and α6β4 exhibit substantial differences in their known signaling functions, it is reasonable to postulate that the unique signaling properties of α6β4 derive largely from the β4 intracellular domain (21Shaw L.M. Rabinovitz I. Wang H.H. Toker A. Mercurio A.M. Cell. 1997; 91: 949-960Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar). Despite its large size, however, the β4 intracellular domain lacks intrinsic kinase activity. A current hypothesis argues that the β4 intracellular domain functions as a “signaling adaptor” that facilitates signaling through growth factor receptors such as Met (22Trusolino L. Bertotti A. Comoglio P.M. Cell. 2001; 107: 643-654Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar). Another viable, though not mutually exclusive, hypothesis is that the association of α6β4 with tetraspanins and its localization in tetraspanin-enriched membrane microdomains enhances its signaling capacity (23Yang X. Kovalenko O.V. Tang W. Claas C. Stipp C.S. Hemler M.E. J. Cell Biol. 2004; 167: 1231-1240Crossref PubMed Scopus (164) Google Scholar). These hypotheses are complicated by the finding that α6β4 signaling can be either dependent on engagement of its ligands (laminins) or independent of such ligation (22Trusolino L. Bertotti A. Comoglio P.M. Cell. 2001; 107: 643-654Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar, 24O'Connor K.L. Shaw L.M. Mercurio A.M. J. Cell Biol. 1998; 143: 1749-1760Crossref PubMed Scopus (136) Google Scholar). One approach to understanding the nature of α6β4 signaling in more detail is to evaluate the intrinsic signaling capacity of the unique β4 intracellular domain itself in the absence of contributions from the α6 subunit and tetraspanins, and to assess the ability of growth factor receptor signaling to cooperate with this intracellular domain. To execute this approach, we generated a chimeric receptor composed of the TrkB extracellular domain and the β4 transmembrane and intracellular domains. Expression of this chimeric receptor in β4-null cancer cells enabled us to assess the signaling potential of the β4 intracellular domain either alone or in response to dimerization of the chimeric receptor using brain-derived neurotrophic factor (BDNF), the ligand for TrkB (25Bibel M. Barde Y.A. Genes Dev. 2000; 14: 2919-2937Crossref PubMed Scopus (863) Google Scholar). The data obtained indicate that the β4 intracellular domain has some intrinsic signaling potential but that it cannot mimic the full signaling capacity of the intact α6β4 integrin. These data also highlight the need to re-evaluate the putative role of the β4 intracellular domain as an “adaptor” for growth factor receptor signaling. Cells—The MDA-MB-435 cancer cell line, which has been reported to be derived from the melanoma cell line M14 (26Rae J.M. Creighton C.J. Meck J.M. Haddad B.R. Johnson M.D. Breast Cancer Res. Treat. 2007; 104: 13-19Crossref PubMed Scopus (289) Google Scholar), was obtained from the Lombardi Breast Cancer Depository at Georgetown University (Washington, D. C.) and maintained in Dulbecco's modified Eagle's medium (DMEM) containing 1 g/liter glucose, 5% fetal bovine serum, 1% penicillin-streptomycin, and 10 mm HEPES. MDA-MB-435 cells stably expressing the α6β4 integrin were described previously (21Shaw L.M. Rabinovitz I. Wang H.H. Toker A. Mercurio A.M. Cell. 1997; 91: 949-960Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar). MDA-MB-435 cells stably expressing full-length TrkB, TrkBextra, and TrkBβ4 were generated as follows. For generation of a full-length TrkB retroviral expression construct, a 2.5-kb EcoRI/SalI fragment was removed from the rat TrkB expression construct pEYFP-N1TrkB (a gift from Dr. Luis Parada, University of Texas Southwestern Medical Center, Dallas, TX), blunt-ended with T4 DNA polymerase (New England Biolabs, Beverly, MA), and subcloned into XhoI-digested and blunt-ended pCLXSN (Imgenex, San Diego, CA). To generate a TrkBextra retroviral expression construct, the extracellular and transmembrane regions (amino acids 1-458) of TrkB were PCR-amplified from pEYFP-N1TrkB with the following primers: Primer 1 (forward) 5′-CCGCTCGAGCGGATGTCGCCCTGGCCGAGGT and Primer 2 (reverse) 5′-CCGCTCGAGCGGCTAATGTCTCGCCAACTTGA. Purified PCR products were then digested with XhoI and subcloned into XhoI-digested pCLXSN. For generation of a retroviral TrkBβ4 expression construct, the TrkB extracellular domain (amino acids 1-429) was first amplified from pEYFP-N1TrkB by PCR, adding 18 bp of sequence complementary to the coding region for the first 6 amino acids of the β4 transmembrane domain (710-715) to the 3′-end of the product, using the following primers: Primer 1 (forward) and Primer 3 (reverse) 5′-GAGCCACCAGAAGGAATGCTCCCGATTGGTT. Next, the transmembrane and intracellular domains of β4 were amplified from pRc/CMVβ4 (27Clarke A.S. Lotz M.M. Chao C. Mercurio A.M. J. Biol. Chem. 1995; 270: 22673-22676Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), adding 18 bp of sequence complementary to the coding region of the final 6 amino acids of the TrkB extracellular domain (424-429) to the 5′-end of the product, using the following primers: Primer 4 (forward) 5′-AACCAATCGGGAGCATTCCTTCTGGTGGCTC and Primer 5 (reverse) 5′-CCGCTCGAGCGGTCAAGTTTGGAAGAACTGT. Finally, these two PCR products were used together as template with Primer 1 (forward) and Primer 5 (reverse) to PCR-amplify the TrkBβ4 insert. This insert was then digested with XhoI and subcloned into pCLXSN. All plasmids were sequenced to confirm that the inserts were correctly inserted and contained no mutations. To generate stable cell lines expressing full-length TrkB, TrkBextra, and TrkBβ4, the above retroviral expression constructs were transfected along with expression plasmids for the vesicular stomatitis virus glycoprotein envelope glycoprotein and Gag-Pol packaging proteins into 293T cells using Lipofectamine 2000 (Invitrogen). Three days post-transfection, viral supernatants were harvested, diluted in serum-containing media supplemented with 8 μg/ml Polybrene (Sigma), and used to infect MDA-MB-435 cells. Following 24 h of infection, cells were selected with Geneticin (2.5 mg/ml) to yield stable cell lines, and cells were then maintained in 1.0 mg/ml Geneticin. Immunoblotting was performed, as described below, to confirm protein expression in the stable cell lines. For the generation of cell lines with stable knockdown of the SHP-2 protein, an shRNA cloned into the pSUPER retroviral expression construct was obtained from Ben Neel (Ontario Cancer Institute, Toronto), and viral production and infection were performed as described above. Puromycin (0.5 μg/ml) was used for cell selection. To create the β4 shRNA-pFSIPPW expression plasmid, the following oligonucleotides (Invitrogen) were annealed and ligated into pFSIPPW (a gift of Andrew Kung, Harvard Medical School, Boston, MA) between the EcoRI and BamHI restriction sites: 5′-aattcccGAGCTGCACGGAGTGTGTCtgcaagagaGACACACTCCGTGCAGCTCtttttg and 5′-gatccaaaaaGAGCTGCACGGAGTGTGTCtctcttgcaGACACACTCGTGCAGCTCggg. To generate stable HCC1937 cell lines expressing β4 shRNA-pFSIPPW the above construct was transfected along with the ViraPower lentiviral packaging mix (Invitrogen) into 293T cells using Lipofectamine 2000 following the manufacturer's instructions. Viral supernatants were harvested 3 days post-transfection, diluted in serum-containing medium supplemented with 8 μg/ml Polybrene, and used to infect HCC1937 cells. Stable cell lines were generated by selection with puromycin (1 μg/ml). Cells were maintained in 0.5 μg/ml puromycin. Suppression of β4 expression was confirmed by immunoblotting as described below. A875 melanoma cells were a generous gift from Alonzo Ross (University of Massachusetts Medical School, Worcester, MA) and were maintained in DMEM containing 1g/liter glucose, 10% fetal bovine serum, 1% penicillin-streptomycin, and 10 mm HEPES. Reverse Transcription-PCR—Total RNA was isolated from cells using an RNeasy mini kit (Qiagen). Gene-specific mRNA was then reverse transcribed into cDNA and amplified from 1 μg of total RNA using a OneStep reverse transcription-PCR kit (Qiagen). PCR amplification of cDNA was performed for 30 cycles using the following primers: p75NTR forward (5′-CGACAACCTCATCCCTGTCT), p75NTR reverse (5′-ACTGCACAGACTCTCCACGA), glyceraldehyde-3-phosphate dehydrogenase forward (5′-ATCACCATCTTCCAGGAGCGA), and glyceraldehyde-3-phosphate dehydrogenase reverse (5′-GCTTCACCACCTTCTTGATGT). Antibodies and Reagents—The following Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA): polyclonal TrkB Ab (H-181), SHP-2 mAb (B-1), phospho-tyrosine mAb (PY99), Src mAb (B-12), and actin Ab (C-11). Abs specific for phospho-SHP-2 (Tyr-542), phospho-Akt (Ser-473), Akt, phospho-p44/42 Erk kinase (Thr-202/Tyr-204), and p44/42 Erk kinase were obtained from Cell Signaling Technology (Beverly, MA). The Src (pTyr-418) and (pTyr-529) Abs were purchased from Invitrogen/BIOSOURCE (Carlsbad, CA). The 505 Ab, raised against a peptide comprising the final 20 amino acids of the β4 C terminus (14) and α6 mAb (2B7) (28Shaw L.M. Lotz M.M. Mercurio A.M. J. Biol. Chem. 1993; 268: 11401-11408Abstract Full Text PDF PubMed Google Scholar) were produced by our laboratory. An mAb against the extracellular domain of β4 (UMA9) was purchased from Ancell (Bayport, MN). Horseradish peroxidase-conjugated anti-rabbit and anti-mouse Abs (Pierce) and a horseradish peroxidase-conjugated anti-goat Ab (Jackson ImmunoResearch, West Grove, PA) were used as secondary Abs. The streptavidin agarose-conjugate, calpeptin, calpastatin peptide, and ALLN were purchased from EMD Biosciences (San Diego, CA), and hepatocyte growth factor (HGF) and BDNF were purchased from Peprotech (Rocky Hill, NJ). Biochemical Analyses—Biotinylation of cell surface proteins was performed using EZ-Link Sulfo-NHS-LC-Biotin (Pierce) following the manufacturer's instructions with a few modifications, as follows. Cells were washed and suspended in PBS (pH 8.0) at a concentration of 25 × 106 cells/ml and labeled with 0.5 mg/ml biotin for 30 min at room temperature. Following the labeling, cells were washed with PBS containing 100 mm glycine to quench and remove excess biotin and then extracted in a Triton X-100 buffer (50 mm Tris-Cl, pH 7.4, 150 mm NaCl, 5 mm EDTA, 1% Triton X-100) supplemented with 1 mm sodium fluoride, 1 mm sodium orthovanadate, and complete-Mini protease inhibitor mixture (Roche Applied Science). For co-immunoprecipitation studies, cells were extracted with either the Triton X-100 buffer or an Nonidet P-40 buffer (50 mm Tris-Cl, pH 8.0, 150 mm NaCl, 5 mm EDTA, 1% Nonidet P-40, 1 mm sodium fluoride, 1 mm sodium orthovanadate, and complete-Mini protease inhibitor mixture). Cell extracts were clarified by centrifugation at 16,000 × g for 10 min and pre-absorbed for 2 h using protein G-Sepharose beads. After centrifugation at 5,000 × g for 5 min to pellet the beads, extracts were incubated with primary Abs for 1 h, and immune complexes were then precipitated with protein G-Sepharose overnight. For precipitation of biotinylated proteins, pre-absorbed extracts were incubated with a streptavidin agarose-conjugate overnight. Precipitates were washed two times with lysis buffer, one time with PBS, and then eluted in 1× reducing SDS sample buffer while boiling for 5 min. For immunoblotting, cell extracts were prepared as described above, and protein concentrations determined using the Bradford assay (Bio-Rad). These extracts, or eluted immune complexes from the immunoprecipitations, were separated by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad). Membranes were then blocked in TBS-T (Tris-Cl, pH 7.5, 150 mm NaCl, 0.05% Tween 20) containing 5% nonfat dry milk for at least 30 min, except for immunoblots for phosphorylated proteins, in which case TBS-T containing 5% BSA was used. Following blocking, membranes were incubated overnight with primary Ab in blocking buffer, washed 3× with TBS-T, incubated with horseradish peroxidase-conjugated secondary Ab in blocking buffer, washed 3× with TBS-T, and detected using SuperSignal West Pico Chemiluminescent substrate (Pierce). For assays where BDNF was used to dimerize the TrkBβ4 chimera, cells were washed once with PBS and serum-deprived for 24 h in DMEM containing 1 g/liter glucose, 1% penicillin-streptomycin, 10 mm HEPES, and 0.1% BSA. Following serum starvation, cells were incubated with fresh serum-free medium supplemented with 100 ng/ml BDNF for the indicated time periods at 37 °C. Treatment of cells with HGF was performed in a similar manner, using HGF at a final concentration of 100 ng/ml. Integrin Clustering—Cells were removed from their dishes with trypsin and washed twice with RPMI medium containing 1% BSA (RH/BSA). After washing, the cells were resuspended in the same buffer at a concentration of 106 cells/ml and incubated for 30 min with either α6- or β4-integrin-specific Abs (2 μg/ml) or in buffer alone. The cells were washed once, resuspended in RH/BSA, and added to plates that had been coated overnight at 4 °C with anti-mouse IgG (100 μg/10-cm plate). The plates were blocked with RH/BSA for 30 min prior to the addition of the cells. Inhibitors were added to the cells for 15 min prior to plating the cells in the antibody-coated plates. After incubation at 37 °C for 15-60 min, the cells were washed twice with PBS and solubilized at 4 °C for 10 min in the Nonidet P-40 lysis buffer. Nuclei were removed by centrifugation at 12,000 × g for 8 min. Migration Assays—Migration assays were performed using Corning (Corning, NY) Transwell chambers (8.0-μm pore size). Membranes were prepared by coating the upper and lower surfaces with 15 μg/ml collagen (Cohesion, Palo Alto, CA) overnight at 4 °C, and then blocking with DMEM containing 0.25% heat-inactivated BSA for 1 h at 37 °C. Cells were then trypsinized, counted, and resuspended in DMEM containing 0.25% heat-inactivated BSA. A total of 1 × 106 cells was added to the upper chamber of the Transwell, and HGF (50 μg/ml) was added to the bottom wells as a chemoattractant. Migration was allowed to proceed for 2.5 h at 37 °C at which time non-migrating cells were removed mechanically from the upper chamber using a cotton swab. Cells that migrated to the lower surface of the Transwell membrane were fixed in methanol for 10 min at room temperature, and membranes were mounted on glass slides using Vectashield mounting medium containing 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA). Migration was quantified by counting the number of stained nuclei in five fields of view in each Transwell, in triplicate. Generation and Characterization of a TrkBβ4 Chimeric Receptor—To assess the intrinsic signaling capacity of the β4 integrin intracellular domain, we generated an expression construct consisting of the extracellular domain of the neuronal TrkB receptor fused to the transmembrane and intracellular domains of β4 (Fig. 1A). We used this system as a model of β4 lateral clustering following adhesion of α6β4 to its extracellular matrix ligand by inducing dimerization of these chimeric molecules with the TrkB ligand, BDNF. We also generated a truncated, signaling incompetent TrkB construct consisting of only the extracellular and transmembrane regions of TrkB to use as a negative control (Fig. 1A). This control enabled us to discount the possibility that endogenous receptors mediate BDNF signaling. These constructs, as well as full-length TrkB, were expressed in MDA-MB-435 cancer cells, which express neither the α6β4 integrin nor the TrkB receptor, as shown in immunoblots of extracts from subclones expressing backbone vector (pCLXSN) alone (Fig. 1B). We chose these cells because they have been used in numerous studies of β4 function and signaling, allowing us to compare the signaling capability of the TrkBβ4 chimera to that of wild-type α6β4 (12Jauliac S. Lopez-Rodriguez C. Shaw L.M. Brown L.F. Rao A. Toker A. Nat. Cell Biol. 2002; 4: 540-544Crossref PubMed Scopus (347) Google Scholar, 21Shaw L.M. Rabinovitz I. Wang H.H. Toker A. Mercurio A.M. Cell. 1997; 91: 949-960Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar, 22Trusolino L. Bertotti A. Comoglio P.M. Cell. 2001; 107: 643-654Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar, 29Bertotti A. Comoglio P.M. Trusolino L. J. Cell Biol. 2006; 175: 993-1003Crossref PubMed Scopus (95) Google Scholar, 30Chen M. O'Connor K.L. Oncogene. 2005; 24: 5125-5130Crossref PubMed Scopus (96) Google Scholar). Expression of the transfected proteins in stable clones was confirmed by immunoblotting using an Ab against the extracellular domain of TrkB (anti-TrkB), which recognizes all three proteins, and one against the intracellular domain (carboxyl 20 amino acids) of β4 (505), which recognizes only the TrkBβ4 chimera (Fig. 1B). To confirm cell surface localization of these expressed proteins, we performed biotinylation followed by immunoprecipitation with streptavidin-agarose and immunoblotting for TrkB. As shown in Fig. 1C, the TrkBβ4 chimera, as well as the full-length TrkB and TrkBextra proteins, were all immunoprecipitated, indicating that they are localized at the cell surface. The TrkBβ4 chimera, unlike the intact α6β4 integrin, should not associate with the α6 subunit, because the association of the α and β subunits of integrin heterodimers occurs through their extracellular domains (31Hynes R.O. Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6687) Google Scholar). Indeed, as shown in Fig. 1D, TrkBβ4 was not detected by immunoblotting in α6 immunoprecipitates, although the full-length β4 subunit was detected. Moreover, we were unable to detect any association of the TrkBβ4 chimera with tetraspanins (data not shown), a result that is consistent with the observation that this association occurs though the α6 subunit of α6β4 (32Sterk L.M. Geuijen C.A. Oomen L.C. Calafat J. Janssen H. Sonnenberg A. J. Cell Biol. 2000; 149: 969-982Crossref PubMed Scopus (189) Google Scholar, 33Yauch R.L. Kazarov A.R. Desai B. Lee R.T. Hemler M.E. J. Biol. Chem. 2000; 275: 9230-9238Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). The neurotrophin, BDNF, not only binds to the TrkB receptor but also binds to the non-selective p75 neurotrophic receptor (p75NTR) with a lower affinity (34Barbacid M. Curr. Opin. Cell Biol. 1995; 7: 148-155Crossref PubMed Scopus (526) Google Scholar). Expression of p75NTR has been observed in several non-neuronal cell types and cancers (35Kruttgen A. Schneider I. Weis J. Brain Pathol. 2006; 16: 304-310Crossref PubMed Scopus (78) Google Scholar, 36Sariola H. Cell Mol. Life Sci. 2001; 58: 1061-1066Crossref PubMed Scopus (108) Google Scholar), including melanoma cells (37Puma P. Buxser S.E. Watson L. Kelleher D.J. Johnson G.L. J. Biol. Chem. 1983; 258: 3370-3375Abstract Full Text PDF PubMed Google Scholar). Given that the MDA-MB-435 cell line may possess properties of melanoma cells (26Rae J.M. Creighton C.J. Meck J.M. Haddad B.R. Johnson M.D. Breast Cancer Res. Treat. 2007; 104: 13-19Crossref PubMed Scopus (289) Google Scholar), we examined whether these cells express p75NTR, to exclude the possibility of signaling through p75NTR following BDNF treatment. Expression of p75NTR mRNA was examined by reverse transcription-PCR. Although the melanoma cell line, A875, expresses a significant amount of mRNA for p75NTR, the MDA-MB-435 TrkBβ4 cell line did not express mRNA for this receptor (Fig. 1E). Dimerization of the β4 Intracellular Domain Induces SHP-2 Binding and Activation—A recent report demonstrated that signaling through the Met receptor can promote the association of the tyrosine phosphatase SHP-2 with the β4 intracellular domain (29Bertotti A. Comoglio P.M. Trusolino L. J. Cell Biol. 2006; 175: 993-1003Crossref PubMed Scopus (95) Google Scholar). A key issue that has not been addressed, however, is whether clustering of the β4 intracellular domain by itself is sufficient to induce SHP-2 binding in the absence of growth factor stimulation. The TrkBβ4 chimera provided an ideal model system for testing this issue. SHP-2 was immunoprecipitated from MDA-MB-435 cells expressing TrkBβ4, and the presence of TrkBβ4 in these immunoprecipitates was assessed by immunoblotting. As shown in Fig. 2A, BDNF stimulation resulted in a significant increase in the amount of TrkBβ4 that co-immunoprecipitated with SHP-2. To verify this finding, we performed the reverse immunoprecipitation to de" @default.
• W2010423785 created "2016-06-24" @default.
• W2010423785 creator A5010527699 @default.
• W2010423785 creator A5023136222 @default.
• W2010423785 creator A5065507815 @default.
• W2010423785 creator A5079389968 @default.
• W2010423785 creator A5082544334 @default.
• W2010423785 date "2007-10-01" @default.
• W2010423785 modified "2023-10-02" @default.
• W2010423785 title "Intrinsic Signaling Functions of the β4 Integrin Intracellular Domain" @default.
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