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• W2058647684 abstract "Nerve growth factor (NGF) binding to its receptors TrkA and p75NTR enhances the survival, differentiation, and maintenance of neurons. Recent studies have suggested that NGF receptor activation may occur in caveolae or caveolae-like membranes (CLM). This is an intriguing possibility because caveolae have been shown to contain many of the signaling intermediates in the TrkA signaling cascade. To examine the membrane localization of TrkA and p75NTR, we isolated caveolae from 3T3-TrkA-p75 cells and CLM from PC12 cells. Immunoblot analysis showed that TrkA and p75NTR were enriched about 13- and 25-fold, respectively, in caveolae and CLM. Binding and cross-linking studies demonstrated that the NGF binding to both TrkA and p75NTRwas considerably enriched in CLM and that about 90% of high affinity binding to TrkA was present in CLM. When PC12 cells were treated with NGF, virtually all activated (i.e. tyrosine phosphorylated) TrkA was found in the CLM. Remarkably, in NGF-treated cells, it was only in CLM that activated TrkA was coimmunoprecipitated with phosphorylated Shc and PLCγ. These results document a signaling role for TrkA in CLM and suggest that both TrkA and p75NTRsignaling are initiated from these membranes. Nerve growth factor (NGF) binding to its receptors TrkA and p75NTR enhances the survival, differentiation, and maintenance of neurons. Recent studies have suggested that NGF receptor activation may occur in caveolae or caveolae-like membranes (CLM). This is an intriguing possibility because caveolae have been shown to contain many of the signaling intermediates in the TrkA signaling cascade. To examine the membrane localization of TrkA and p75NTR, we isolated caveolae from 3T3-TrkA-p75 cells and CLM from PC12 cells. Immunoblot analysis showed that TrkA and p75NTR were enriched about 13- and 25-fold, respectively, in caveolae and CLM. Binding and cross-linking studies demonstrated that the NGF binding to both TrkA and p75NTRwas considerably enriched in CLM and that about 90% of high affinity binding to TrkA was present in CLM. When PC12 cells were treated with NGF, virtually all activated (i.e. tyrosine phosphorylated) TrkA was found in the CLM. Remarkably, in NGF-treated cells, it was only in CLM that activated TrkA was coimmunoprecipitated with phosphorylated Shc and PLCγ. These results document a signaling role for TrkA in CLM and suggest that both TrkA and p75NTRsignaling are initiated from these membranes. nerve growth factor epidermal growth factor EGF receptor phospholipase-C-γ-1 mitogen-activated protein kinase caveolae-like membranes noncaveolae-like membranes phosphate-buffered saline polyacrylamide gel electrophoresis platelet-derived growth factor interleukin The neurotrophins make up a family of structurally related polypeptide neurotrophic factors that bind to specific receptors to enhance the survival, differentiation, and maintenance of neurons in both the central and peripheral nervous system (1Levi-Montalcini R. Science. 1987; 237: 1154-1162Crossref PubMed Scopus (2658) Google Scholar, 2Theonen H. Trends Neurosci. 1991; 14: 165-170Abstract Full Text PDF PubMed Scopus (758) Google Scholar, 3Zang L. Schmidt R.E. Yan Q. Snider W.D. J. Neurosci. 1994; 14: 5187-5201Crossref PubMed Google Scholar, 4Yuen E.C. Mobley W.C. Ann. Neurol. 1996; 40: 346-354Crossref PubMed Scopus (143) Google Scholar). Nerve growth factor (NGF)1 is the best characterized neurotrophin, and much is known about NGF structure and actions (4Yuen E.C. Mobley W.C. Ann. Neurol. 1996; 40: 346-354Crossref PubMed Scopus (143) Google Scholar, 5Orlinick J.R. Chao M.V. Cell Signal. 1998; 10: 543-551Crossref PubMed Scopus (72) Google Scholar, 6Connor B. Dragunow M. Brain Res. Brain Res. Rev. 1998; 27: 1-39Crossref PubMed Scopus (475) Google Scholar, 7Longo F.M. Holtzman D.M. Grimes M.L. Mobley W.C. Fallon J. Loughlin S. Neurotrophic Factors. Academic Press, New York1993: 209-256Google Scholar). However, important questions remain with respect to the signaling properties and membrane trafficking of NGF receptors. Two distinct classes of binding sites differ in their affinity for NGF by 2 orders of magnitude (8Sutter A. Riopelle R.J. Harris-Warrick R.M. Shooter E.M. J. Biol. Chem. 1979; 254: 5972-5982Abstract Full Text PDF PubMed Google Scholar, 9Meakin S.O. Shooter E.M. Trends Neurosci. 1992; 15: 323-331Abstract Full Text PDF PubMed Scopus (576) Google Scholar). Low affinity (10−9m) and high affinity (10−11m) NGF receptors are distinguished kinetically by a much slower rate of dissociation from the high affinity sites (9Meakin S.O. Shooter E.M. Trends Neurosci. 1992; 15: 323-331Abstract Full Text PDF PubMed Scopus (576) Google Scholar, 10Landreth G.E. Shooter E.M. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 4751-4755Crossref PubMed Scopus (152) Google Scholar, 11Schechter A.L. Bothwell M.A. Cell. 1981; 24: 867-874Abstract Full Text PDF PubMed Scopus (179) Google Scholar, 12Woodruff N.R. Neet K.E. Biochemistry. 1986; 25: 7956-7966Crossref PubMed Scopus (38) Google Scholar). Many NGF actions in neurons appear to be mediated by high affinity receptors (9Meakin S.O. Shooter E.M. Trends Neurosci. 1992; 15: 323-331Abstract Full Text PDF PubMed Scopus (576) Google Scholar). NGF binds to two receptor proteins, TrkA and p75NTR (13Kaplan D.R. Miller F.D. Curr. Opin. Cell Biol. 1997; 9: 213-221Crossref PubMed Scopus (542) Google Scholar). TrkA is a receptor tyrosine kinase whose activation accounts for many of the classical neurotrophic properties of NGF. NGF binding induces TrkA dimerization, kinase activation, and receptor tyrosine autophosphorylation (14Kaplan D.R. Martin-Zanca D. Parada L.F. Nature. 1991; 350: 158-160Crossref PubMed Scopus (831) Google Scholar, 15Kaplan D.R. Hempstead B.L. Martin-Zanca D. Chao M.V. Parada L.F. Science. 1991; 252: 554-558Crossref PubMed Scopus (1131) Google Scholar, 16Klein R. Jing S.Q. Nanduri V. O'Rourke E. Barbacid M. Cell. 1991; 65: 189-197Abstract Full Text PDF PubMed Scopus (1132) Google Scholar, 17Meakin S.O. Shooter E.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5862-5866Crossref PubMed Scopus (46) Google Scholar, 18Jing S. Tapley P. Barbacid M. Neuron. 1992; 9: 1067-1079Abstract Full Text PDF PubMed Scopus (385) Google Scholar). Dissociation of NGF from TrkA is slow (19Meakin S.O. Suter U. Drinkwater C.C. Welcher A.A. Shooter E.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2374-2378Crossref PubMed Scopus (185) Google Scholar), suggesting that TrkA contributes to the formation of high affinity receptors. However, TrkA alone is unlikely to account for high affinity NGF binding because most binding to TrkA in cells expressing only this receptor is of low affinity (20Mahadeo D. Kaplan L. Chao M.V. Hempstead B.L. J. Biol. Chem. 1994; 269: 6884-6891Abstract Full Text PDF PubMed Google Scholar). p75NTR is a single-transmembrane glycoprotein; it is a member of the TNF receptor superfamily (21Carter B.D. Lewin G.R. Neuron. 1997; 18: 187-190Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). p75NTR binds with low affinity to NGF and other members of the neurotrophin family (22Rodriguez-Tebar A. Dechant G. Gotz R. Barde Y.A. EMBO J. 1992; 11: 917-922Crossref PubMed Scopus (383) Google Scholar). p75NTRappears to signal independently of TrkA as well as to regulate TrkA signaling (21Carter B.D. Lewin G.R. Neuron. 1997; 18: 187-190Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 23Dobrowsky R.T. Werner M.H. Castellino A.M. Chao M.V. Hannun Y.A. Science. 1994; 265: 1596-1599Crossref PubMed Scopus (548) Google Scholar, 24Dobrowsky R.T. Jenkins G.M. Hannun Y.A. J. Biol. Chem. 1995; 270: 22135-22142Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 25Cassaccia-Bonnefil P. Carter B.D. Dobrowsky R.T. Chao M.V. Nature. 1996; 383: 716-719Crossref PubMed Scopus (712) Google Scholar, 26Carter B.D. Kaltschmidt B. Offenhauser N. Bohm-Matthaei R. Baeuerle P.A. Barde YA. Science. 1996; 272: 542-545Crossref PubMed Scopus (610) Google Scholar, 27Chao V.M. Hempstead B.L. Trends Neurosci. 1995; 18: 321-326Abstract Full Text PDF PubMed Scopus (764) Google Scholar). The latter may be due, in part, to direct interaction of p75NTR with TrkA. p75NTR has been shown to increase the rate of association of NGF with TrkA, thereby increasing TrkA activation and the number of high affinity binding sites (20Mahadeo D. Kaplan L. Chao M.V. Hempstead B.L. J. Biol. Chem. 1994; 269: 6884-6891Abstract Full Text PDF PubMed Google Scholar, 28Verdi J.M. Birren D.J. Ibanez C.F. Persson H. Kaplan D.R. Benedetti M. Chao M.V. Anderson D.J. Neuron. 1994; 12: 733-745Abstract Full Text PDF PubMed Scopus (311) Google Scholar). Moreover, there is increasing evidence from photobleaching studies (29Wolf D.E. McKinnon C.A. Daou M.C. Stephens R.M. Kaplan D.R. Ross A.H. J. Biol. Chem. 1995; 270: 2133-2138Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), copatching experiments (30Ross A.H. Daou M.C. McKinnon C.A. Condon P.J. Lachyankar M.B. Stephens R.M. Kaplan D.R. Wolf D.E. J. Cell Biol. 1996; 132: 945-953Crossref PubMed Scopus (70) Google Scholar), cross-linking (31Ross G.M Shamovsky I.L Lawrance G. Solc M. Dostaler S.M. Weaver D.F. Riopelle R.J. Eur. J. Neurosci. 1998; 10: 890-898Crossref PubMed Scopus (70) Google Scholar), and co-immunoprecipitation studies (32Gargano N. Levi A. Alema S. J. Neurosci. Res. 1997; 50: 1-12Crossref PubMed Scopus (57) Google Scholar, 33Huber L.J. Chao M.V. J. Neurosci. Res. 1995; 40: 557-563Crossref PubMed Scopus (119) Google Scholar, 34Bibel M. Hoppe E. Barde Y.-A. EMBO J. 1999; 18: 616-622Crossref PubMed Scopus (369) Google Scholar) to suggest that p75NTR and TrkA interact directly in surface membranes. Caveolae are specialized membrane microdomains that in many cells exist as vesicular invaginations of the plasma membrane (35Schlegel A. Volonte D. Engelman J.A. Galbiati F. Mehta P. Zang X.L. Scherer P.E. Lisanti M.P. Cell Signal. 1998; 10: 457-463Crossref PubMed Scopus (150) Google Scholar, 36Anderson R.G.W. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1711) Google Scholar, 37Brown D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Crossref PubMed Scopus (2529) Google Scholar). Enriched in cholesterol, sphingolipids, and the ganglioside GM1, caveolae contain structural proteins of the caveolin family (36Anderson R.G.W. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1711) Google Scholar, 38Okamato T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1336) Google Scholar). In cell homogenates, caveolae sediment with a low buoyant density (36Anderson R.G.W. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1711) Google Scholar, 39Smart E.J. Ying Y.-S. Mineo C. Anderson R.G.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10104-10108Crossref PubMed Scopus (671) Google Scholar). Membranes of similar composition and properties have been isolated from cells that do not contain caveolin; in these cells these membranes have been referred to as CLMs (40Wu C. Butz S. Ying Y.-S. Anderson R.G.W. J. Biol. Chem. 1997; 272: 3554-3559Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Wu et al. (40Wu C. Butz S. Ying Y.-S. Anderson R.G.W. J. Biol. Chem. 1997; 272: 3554-3559Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar) showed that TrkB and p75NTR were highly enriched in membranes of low buoyant density prepared from the synaptic plasma membranes of rat forebrain. p75NTR was also enriched in caveolae in p75NTR-transfected 3T3 cells and in CLMs in PC12 cells (41Bilderback T.R. Grigsby R.J. Dobrowsky R.T. J. Biol. Chem. 1997; 272: 10922-10927Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). In addition, many of the intermediates in the TrkA signaling cascade are also present in caveolae or CLMs, including phosphatidylinositol 3-kinase, phospholipase C-γ, Shc, Grb2, SOS-1, Ras, Raf-1, and MAPK (ERK2) (36Anderson R.G.W. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1711) Google Scholar, 42Liu P. Ying Y. Ko Y.-G. Anderson R.G.W. J. Biol. Chem. 1996; 271: 10299-10303Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 43Mineo C. James G.L. Smart E.J. Anderson R.G.W. J. Biol. Chem. 1996; 271: 11930-11935Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar, 44Song K.S. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (915) Google Scholar, 45Liu J. Oh P. Horner T. Rogers R.A. Schnitzer J.E. J. Biol. Chem. 1997; 272: 7211-7222Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). The possibility that caveolae or CLMs could serve as an important locus of NGF receptor binding and signaling prompted us to examine the distribution of TrkA and p75NTR in cells expressing these receptors. We show that both TrkA and p75NTR are enriched in caveolae and CLMs, that NGF binds with high affinity to its receptors in CLMs, and that TrkA activation and signaling are initiated in these membranes. RTA antibody, a polyclonal antibody raised against the extracellular domain of rat TrkA, was a gift of Dr. L. F. Reichardt (University of California, San Francisco) (30Ross A.H. Daou M.C. McKinnon C.A. Condon P.J. Lachyankar M.B. Stephens R.M. Kaplan D.R. Wolf D.E. J. Cell Biol. 1996; 132: 945-953Crossref PubMed Scopus (70) Google Scholar). Anti-phosphotyrosine, anti-phospholipase Cγ-1, anti-EGFR, anti-TrkA ED, and anti-Shc antibodies were purchased from Upstate Biotechnology (Lake Placid, NY). Anti-TrkA (C terminus), anti-Ras, and anti-caveolin-1 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The rabbit polyclonal antibody to caveolin-1 used for ultrastructural studies was obtained from Transduction Laboratories (Lexington, KY). Mouse NGF was prepared by ion-exchange chromotography as indicated (46Zhou J. Holtzman D.M. Weiner R.I. Mobley W.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3824-3828Crossref PubMed Scopus (31) Google Scholar). Unless specified, all other reagents were purchased from Sigma. PC12 cells were maintained in Dulbecco's modified Eagle's medium (DME H-21) supplemented with 10% heat-inactivated horse serum and 5% fetal calf serum. 3T3-TrkA cells were established by transfecting NIH-3T3 cells with a rattrkA expression vector, pDM115 (a gift of Dr. M. V. Chao, New York University Medical Center) (46Zhou J. Holtzman D.M. Weiner R.I. Mobley W.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3824-3828Crossref PubMed Scopus (31) Google Scholar). To establish 3T3-p75 cells, the plasmid NGFR1 (a gift of Dr. E. M. Shooter, Stanford University) was cut with NcoI, blunt-ended, and digested with BglII. The resulting 1.5-kilobase pair DNA fragment, which contains the full-length open reading frame for p75NTR cDNA, was inserted into pREP4 expression vector (Invitrogen, Carlsbad, CA). This p75NTR expression plasmid (pMHZ100) was used to transfect NIH-3T3 cells. 3T3 cells that co-express TrkA and p75NTR (3T3-TrkA-p75) were established by co-transfecting the cells with both pDM115 and pMHZ100. The parental 3T3 cells and their derivatives were grown in DME H-21 medium containing 10% bovine calf serum and appropriate antibiotics (500 μg/ml G418 or 200 μg/ml hygromycin or both). The cells were treated with NGF, or with vehicle alone, at 37 °C for the time indicated. The treated cells were washed with ice-cold PBS. Cells were fixed for immunoelectron microscopy by adding to the culture medium an equal volume of freshly prepared 4% formaldehyde in 0.1 mphosphate buffer, pH 7.4. After 30 min at room temperature, the medium plus fixative was replaced by 4% formaldehyde, and cells were post-fixed for at least 24 h at 4 °C. Fixed cells were washed three times with 0.15% glycine in phosphate buffer, and gently scraped from the dish in a small volume of 1% gelatin. Cells were pelleted in 10% gelatin and, after solidification of the gelatin on ice, cut into ∼1-mm3 blocks. Further preparation for ultrathin cryosectioning and immunogold labeling was carried out as described (47Slot J.W. Geuze H.J. Gigengack S. Lienhard G.E. James J.E. J. Cell Biol. 1991; 113: 123-135Crossref PubMed Scopus (704) Google Scholar, 48Liou W. Geuze H.J. Slot J.W. Histochem. Cell Biol. 1996; 106: 41-58Crossref PubMed Scopus (434) Google Scholar). Caveolae and CLM were prepared using a nondetergent extraction method essentially as described (41Bilderback T.R. Grigsby R.J. Dobrowsky R.T. J. Biol. Chem. 1997; 272: 10922-10927Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 44Song K.S. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (915) Google Scholar). PC12 cells or 3T3 cells and their derivatives were grown to confluence in 15-cm tissue culture dishes; three dishes of cells were used for each preparation. After two washes with ice-cold PBS, all procedures were carried out at 4 °C. The cells were scraped into 2 ml of 0.5 m sodium carbonate buffer, pH 11.0. The cells were homogenized sequentially using a loose fitting Dounce homogenizer (10 strokes), a Polytron tissue grinder (three 10-s bursts), and a sonicator (3 × 20-s bursts). The homogenate (2 ml) was then adjusted to 45% w/v sucrose by adding 2 ml of 90% w/v sucrose prepared in 2× MBS buffer (MBS is 25 mm Mes, pH 6.5, 150 mm NaCl). The final pH of the mixture was 10.2. A discontinuous sucrose gradient was formed by overlaying this solution with 4 ml of 35% w/v sucrose and 4 ml of 5% w/v sucrose; both in MBS containing 250 mm sodium carbonate. The samples were centrifuged at 39,000 rpm in a SW41 rotor for 16–20 h. From the top of the gradient, 0.85-ml gradient fractions were collected to yield a total of 14 fractions. In some experiments, the individual gradient fractions were pooled into CLM (fractions 4–7) and NCM (fractions 10–14). The membranes from each pooled gradient fractions were concentrated by centrifugation at 100,000 × g for 2 h at 4 °C in MBS buffer. The resulting membrane fractions were resuspended in lysis buffer and used for immunoprecipitation or Western blotting analysis. The pooled membrane fractions were resuspended in 0.8 ml of lysis buffer (10 mm Tris-HCl, pH 7.4, 10 mm EDTA, 1% Nonidet P-40, 0.4% deoxycholate, 60 mm β-octylglucoside, 1 mmphenylmethylsulfonyl fluoride, 1 mm sodium orthovanadate, 0.1 mg/ml each of leupeptin and aprotinin). After sonication (3 × 20 s), insoluble material was removed by centrifugation at 4 °C (14,000 × g for 2 min). The lysate was then mixed with antibodies at 4 °C overnight. The immunoprecipitate was collected by incubating with protein A-Sepharose (Pierce) for 2 h. The beads were washed with 3 × 1 ml of lysis buffer followed by 1 ml of PBS. The bound proteins were solubilized in SDS-PAGE sample buffer. Samples were analyzed using 4–15% SDS-PAGE (49Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205496) Google Scholar). The separated proteins were transferred to a nitrocellulose filter. Blots were blocked with 2% bovine serum albumin in TBST (20 mm Tris-HCl, pH 8.0, 150 mm NaCl, 0.2% Tween-20) for 1 h. The membrane was first probed for 3 h with primary antibodies at the dilution suggested by the suppliers. After washing three times with TBST, the blots were then incubated for 1 h with goat anti-rabbit IgGs, goat anti-mouse IgGs, or protein A (for probing caveolin), each conjugated to horseradish peroxidase (1:10,000). The membranes were then washed three times with TBST. The SuperSignal (Pierce) was used to visualize the reaction. For detecting GM1, the nitrocellulose was probed with cholera toxin B-horseradish peroxidase conjugate (1:2000) and washed three times with TBST, and the bound conjugate was visualized with SuperSignal. NGF binding and cross-linking was carried out essentially as described (46Zhou J. Holtzman D.M. Weiner R.I. Mobley W.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3824-3828Crossref PubMed Scopus (31) Google Scholar). PC12 cells (107/15-cm dish) were washed with binding buffer (PBS containing 1 mg/ml each of glucose and bovine serum albumin) at 4 °C for 10 min and then incubated with 2 ml of binding buffer containing 2 nm125I-NGF for 1 h at 4 °C. To measure binding to high affinity (slowly dissociating) receptors, cells were washed with ligand-free binding buffer for 1 h at 4 °C before cross-linking. To correct for nonspecific binding and cross-linking, unlabeled NGF (1 μm) was included during binding and cross-linking. To cross-link NGF to TrkA, bis-sulfosuccinimidyl suberate was added to a final concentration of 0.8 mm in binding buffer. For cross-linking NGF to p75NTR, EDAC was used at a final concentration of 20 mm. Reaction mixtures were incubated at 4 °C for 30 min, followed by washing cells with binding buffer. The cross-linking reaction was stopped by adding 10 mm of glutathione to the samples. The samples were then subjected to membrane fractionation as described above. The resulting fractions were pooled as described, and the proteins were separated using 4–15% SDS-PAGE. Gels were then fixed, dried, and exposed to x-ray film XAR-5 (Eastman Kodak Co.). The film was scanned by a Umax superVista S-12 scanner, and intensity of the each protein band was measured using NIH image software. Biotinylation of surface receptors was carried out as described (50Grimes M.L. Zhou J. Beattie E.C. Yuen E.C. Hall D.E. Valletta J.S. Topp K.S. LaVail J.H. Bunnet N.W. Mobley W.C. J. Neurosci. 1996; 16: 7950-7964Crossref PubMed Google Scholar). PC12 cells were incubated with 0.5 mg/ml of NHS-SS-Biotin (Pierce) in PBS at 4 °C for 30 min. The reaction was terminated by washing followed by adding lysine (1 mm) to react with remaining biotin. The CLM and NCM fractions were prepared as described above. To show whether or not p75NTR and TrkA are present in caveolae, we used nondetergent extraction to isolate caveolae from 3T3-TrkA-p75 cells. Fourteen fractions (0.85 ml each) from the discontinuous sucrose gradient were collected and analyzed for distribution of p75NTR, TrkA, and caveolar markers including the proteins caveolin-1, Ras and the ganglioside GM-1. As expected from earlier studies, caveolin-1, Ras, and GM-1 were found almost exclusively in fractions 4–6 (Fig. 1). Immunoblot analysis showed that about 40% of TrkA and 60% of p75NTRwere also present in fractions 4–6. The ratio of TrkA in caveolar to noncaveolar fractions was 0.6; the caveolar to noncaveolar ratio for p75NTR was 1.5. Because the amount of protein in these fractions accounts for only about 5% of total cellular protein, TrkA was enriched 13-fold in these membranes, whereas p75NTR was enriched 25-fold. The distribution of TrkA was similar in 3T3-TrkA cells (data not shown). Using immunoprecipitation, some receptors and signaling proteins localized to caveolae can be shown to interact with caveolin-1. In agreement with an earlier study (37Brown D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Crossref PubMed Scopus (2529) Google Scholar), p75NTR was found in anti-caveolin-1 immunoprecipitates from 3T3-TrkA-p75 cells (Fig. 2 A). NGF treatment had no effect on the ability to coimmunoprecipitate p75NTR with caveolin, suggesting that NGF binding to p75NTR has no effect on the interaction of p75NTR with caveolin-1. In the same experiments, we found no evidence for immunoprecipitation of TrkA with anti-caveolin-1 antibodies (Fig. 2 A). This suggests that TrkA either fails to interact with caveolin-1 or does so with lesser affinity than p75NTR. Palmitoylation marks many proteins present in caveolae (51Barker P.A. Barbee G. Misko T.P. Shooter E.M. J. Biol. Chem. 1994; 269: 30645-30650Abstract Full Text PDF PubMed Google Scholar, 52Dietzen D.J. Hastings W.R. Lublin D.M. J. Biol. Chem. 1995; 270: 6838-6842Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar), and for some proteins this modification enhances their targeting to this locus (53Shenoy-Scaria A.M. Dietzen D.J. Kwong J. Link D.C. Lublin D.M. J. Cell Biol. 1994; 126: 353-363Crossref PubMed Scopus (340) Google Scholar). p75NTR was shown to be palmitoylated in an earlier study (51Barker P.A. Barbee G. Misko T.P. Shooter E.M. J. Biol. Chem. 1994; 269: 30645-30650Abstract Full Text PDF PubMed Google Scholar). To ask whether TrkA was also palmitoylated, we incubated 3T3-TrkA cells with [3H]palmitic acid, and, after lysis, determined whether the receptor was acylated. SDS-PAGE analysis of TrkA immunoprecipitates showed that TrkA was covalently linked to the fatty acid (Fig. 2 B). This covalent bound was sensitive to 2-mercaptoethanol, indicating that like other palmitoylated proteins found in caveolae (51Barker P.A. Barbee G. Misko T.P. Shooter E.M. J. Biol. Chem. 1994; 269: 30645-30650Abstract Full Text PDF PubMed Google Scholar, 52Dietzen D.J. Hastings W.R. Lublin D.M. J. Biol. Chem. 1995; 270: 6838-6842Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar, 53Shenoy-Scaria A.M. Dietzen D.J. Kwong J. Link D.C. Lublin D.M. J. Cell Biol. 1994; 126: 353-363Crossref PubMed Scopus (340) Google Scholar), palmitic acid is linked through a thioester bond to a cysteine residue(s) in TrkA. Palmitoylation of TrkA and p75NTR is consistent with their presence in caveolae. Filipin binds cholesterol and disrupts caveolae (54Schnitzer J.E. Oh P. Pinney E. Allard J. J. Cell Biol. 1994; 127: 1217-1232Crossref PubMed Scopus (765) Google Scholar). Filipin treatment has been shown to decrease PDGF-mediated activation of PDGF receptor, a receptor tyrosine kinase localized in caveolae, and to decrease signaling downstream from PDGF receptor (41Bilderback T.R. Grigsby R.J. Dobrowsky R.T. J. Biol. Chem. 1997; 272: 10922-10927Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). To show whether or not filipin would also reduce TrkA activation, 3T3-TrkA cells were treated with filipin (10 μg/ml) for 30 min prior to adding NGF (5 ng/ml). TrkA phosphorylation was decreased to about 63% (± 6%) of the nonfilipin treated control (n = 5) (Fig. 2 C). To ask whether altered TrkA signaling following filipin treatment was associated with a change in the distribution of the receptor, we examined gradients prepared from 3T3-TrkA cells treated with filipin under conditions identical to those used to examine signaling. Compared with untreated cells, TrkA was more widely distributed in filipin treated cells. There was an increase in TrkA in the heaviest CLM fraction (i.e. fraction number 6) and in fractions 7–11 (Fig. 2 D). These results show that the distribution and signaling of TrkA receptors is influenced by a treatment that disrupts caveolae. They provide further support for the localization of TrkA in caveolae. Both differentiated and undifferentiated PC12 cells have been reported to express caveolin-1 (55Galbiati F. Volonte D. Gil O. Zanazzi G. Salzer J.L. Sargiacomo M. Scherer P.E. Engelman J.A. Schlegel A. Parenti M. Okamoto T. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10257-10262Crossref PubMed Scopus (159) Google Scholar, 59Bilderback T.R. Gazula V.R. Lisanti M.P. Dobrowsky R.T. J. Biol. Chem. 1999; 274: 257-263Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Using a fractionation protocol that enriches for CLM, a small amount of caveolin was present in immunoprecipitates from undifferentiated PC 12 cells (59Bilderback T.R. Gazula V.R. Lisanti M.P. Dobrowsky R.T. J. Biol. Chem. 1999; 274: 257-263Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). We found very little evidence for caveolin-1 in the PC12 cells used for our experiments. Whereas by immunoprecipitation followed by Western blot we readily detected caveolin-1 in 3T3 cells, we were unable to do so in untreated PC12 cells or in PC12 cells treated with NGF for 4 days (Fig. 3 D). We also carried out immunoelectron microscopy analysis. Membrane invaginations characteristic of caveolae were seen in both 3T3-TrkA and 3T3 cells (Fig. 3, A and B). In both, we found immunolabeling of caveolin-1 in these structures. However, we could not show that caveolin-1 was present in PC12 cells (Fig. 3 C) using conditions under which the protein was readily seen in 3T3 cells. We conclude that there is little, if any, caveolin-1 in the PC12 cells used in our experiments. To determine whether or not the membrane localization of p75NTR and TrkA in PC12 cells is similar to that in 3T3 cells, PC12 cells were subjected to extraction and fractionation as described above. Aliquots (15 μl) of fractions were analyzed by SDS-PAGE and the proteins were visualized using Coomassie Blue staining. About 95% of cellular protein was found in fractions 10–14 (Fig. 4 A). Only 5% was present in fractions 4–6. Immunoblot analysis showed that most GM1 was in factions 5 and 6 (Fig. 4 B), the same fractions in which GM1 was found in 3T3 cells. Immunoblots revealed that Ras was also concentrated in fractions 5 and 6. β3-Integrin, a membrane protein that is excluded from caveolae (39Smart E.J. Ying Y.-S. Mineo C. Anderson R.G.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10104-10108Crossref PubMed Scopus (671) Google Scholar), was found only in fractions at the bottom of the sucrose gradient (fractions 10–14). These results indicate that PC12 cells contain membrane domains similar to caveolae in buoyant density, ganglioside content, and protein constituents. In accord with others (40Wu C. Butz S. Ying Y.-S. Anderson R.G.W. J. Biol. Chem. 1997; 272: 3554-3559Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar), we refer to them as CLM. We designated fractions 4–6 as CLM and 10–14 as NCM. To show whether or not TrkA and p75NTR were present in CLM, an aliquot of each gradient fraction was analyzed using antibodies to TrkA or p75NTR. Bands of the correct m" @default.
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• W2058647684 date "1999-12-01" @default.
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• W2058647684 title "Nerve Growth Factor Signaling in Caveolae-like Domains at the Plasma Membrane" @default.
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