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• W2089124684 abstract "Ligand-induced receptor oligomerization is an established mechanism for receptor-tyrosine kinase activation. However, numerous receptor-tyrosine kinases are expressed in multicomponent complexes with other receptors that may signal independently or alter the binding characteristics of the receptor-tyrosine kinase. Nerve growth factor (NGF) interacts with two structurally unrelated receptors, the Trk A receptor-tyrosine kinase and p75, a tumor necrosis factor receptor family member. Each receptor binds independently to NGF with predominantly low affinity (K d = 10−9m), but they produce high affinity binding sites (K d = 10−11m) upon receptor co-expression. Here we provide evidence that the number of high affinity sites is regulated by the ratio of the two receptors and by specific domains of Trk A and p75. Co-expression of Trk A containing mutant transmembrane or cytoplasmic domains with p75 yielded reduced numbers of high affinity binding sites. Similarly, co-expression of mutant p75 containing altered transmembrane and cytoplasmic domains with Trk A also resulted in predominantly low affinity binding sites. Surprisingly, extracellular domain mutations of p75 that abolished NGF binding still generated high affinity binding with Trk A. These results indicate that the transmembrane and cytoplasmic domains of Trk A and p75 are responsible for high affinity site formation and suggest that p75 alters the conformation of Trk A to generate high affinity NGF binding. Ligand-induced receptor oligomerization is an established mechanism for receptor-tyrosine kinase activation. However, numerous receptor-tyrosine kinases are expressed in multicomponent complexes with other receptors that may signal independently or alter the binding characteristics of the receptor-tyrosine kinase. Nerve growth factor (NGF) interacts with two structurally unrelated receptors, the Trk A receptor-tyrosine kinase and p75, a tumor necrosis factor receptor family member. Each receptor binds independently to NGF with predominantly low affinity (K d = 10−9m), but they produce high affinity binding sites (K d = 10−11m) upon receptor co-expression. Here we provide evidence that the number of high affinity sites is regulated by the ratio of the two receptors and by specific domains of Trk A and p75. Co-expression of Trk A containing mutant transmembrane or cytoplasmic domains with p75 yielded reduced numbers of high affinity binding sites. Similarly, co-expression of mutant p75 containing altered transmembrane and cytoplasmic domains with Trk A also resulted in predominantly low affinity binding sites. Surprisingly, extracellular domain mutations of p75 that abolished NGF binding still generated high affinity binding with Trk A. These results indicate that the transmembrane and cytoplasmic domains of Trk A and p75 are responsible for high affinity site formation and suggest that p75 alters the conformation of Trk A to generate high affinity NGF binding. epidermal growth factor receptor glial-derived neurotrophic growth factor nerve growth factor multiplicity of infection brain-derived neurotrophic factor polyacrylamide gel electrophoresis epidermal growth factor wild type 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide Growth factor receptor-tyrosine kinases, such as the epidermal growth factor receptor (EGFR),1 undergo ligand-induced oligomerization, leading to receptor activation. Although homodimerization of receptor-tyrosine kinases is sufficient to initiate transmembrane signaling, an increasing number of receptor-tyrosine kinases have been found to be co-expressed with heterologous co-receptors lacking intrinsic kinase activity. Examples of such heteromeric receptors include the receptors for vascular endothelial growth factor, the flt-1 receptor-tyrosine kinase and neuropilin-1 (1Fuh G. Garcia K.C. de Vos A.M. J. Biol. Chem. 2000; 275: 26690-26695Abstract Full Text Full Text PDF PubMed Google Scholar); the receptors for the glial-derived neurotrophic growth factor (GDNF), the ret receptor-tyrosine kinase and GRFα (2Eketjall S. Fainzilber M Murray-Rust J. Ibanez C.F. EMBO J. 1999; 18: 5901-5910Crossref PubMed Scopus (106) Google Scholar); and the receptors for nerve growth factor (NGF), the Trk A receptor-tyrosine kinase and the p75 neurotrophin receptor (3Hempstead B.L. Martin-Zanca D. Kaplan D.R. Parada L.F. Chao M.V. Nature. 1991; 350: 678-683Crossref PubMed Scopus (1022) Google Scholar). With the vascular endothelial growth factor and NGF ligands, co-expression of their receptor-tyrosine kinase and heterologous co-receptor generates receptor complexes that exhibit higher affinity binding constants than those exhibited by homodimeric receptor-tyrosine kinase complexes. For GDNF receptor activation, expression of both the ret receptor-tyrosine kinase and GRFα subunit is required for ligand binding because GDNF is unable to activate homodimeric ret receptor complexes (4Trupp M. Raynochek C. Belluardo N. Ibanez C.F. Mol. Cell. Neurosci. 1998; 11: 47-63Crossref PubMed Scopus (162) Google Scholar). Several models have been proposed to accommodate co-operative roles for these dual receptor systems. A model in which the nonreceptor-tyrosine kinase first binds the ligand, alters the local concentration, and then passes the ligand to the receptor-tyrosine kinase has been proposed. A second, conformational model predicts that co-expression of both receptor-tyrosine kinase and co-receptor alters the conformation of the receptor-tyrosine kinase through allosteric interactions, generating a higher affinity binding site by altering the association or dissociation constants of the ligand with its receptor-tyrosine kinase. We have utilized the NGF receptors as a model system to clarify a mechanism that generates high affinity site formation. NGF belongs to the neurotrophin family of survival and differentiation factors, which also includes BDNF, NT-3, and NT-4/5. NGF-responsive neurons exhibit two classes of binding sites based upon equilibrium binding reactions (5Sutter A. Riopelle R.J. Harris-Warrick R.M. Shooter E.M. J. Biol. Chem. 1979; 254: 5972-5982Abstract Full Text PDF PubMed Google Scholar). The transmembrane proteins responsible for high affinity NGF binding (K d 10−11m) are the Trk A receptor and the p75 neurotrophin receptor (which binds all neurotrophins (6Rodriguez-Tebar A. Dechant G. Gotz R. Barde Y.A. EMBO J. 1992; 11: 917-922Crossref PubMed Scopus (385) Google Scholar)). Kinetic analysis of NGF binding indicates that each receptor binds NGF with a relatively low affinity K dbetween 10−9 and 10−10m (7Mahadeo D. Kaplan L. Chao M.V. Hempstead B.L. J. Biol. Chem. 1994; 269: 6884-6891Abstract Full Text PDF PubMed Google Scholar). Although p75 displays very rapid rates of association and dissociation with NGF, the Trk A receptor has much slower on- and off-rates. When Trk A receptors are co-expressed with p75, the rate of association is accelerated 25-fold, generating a new kinetic site exhibiting high affinity binding properties (7Mahadeo D. Kaplan L. Chao M.V. Hempstead B.L. J. Biol. Chem. 1994; 269: 6884-6891Abstract Full Text PDF PubMed Google Scholar). Thus, one function of p75 is to increase the binding affinity of NGF. How this is accomplished has not been determined. Direct interactions between the p75 and Trk A receptors have been difficult to document biochemically, although ligand-induced receptor homodimers can be readily detected in affinity cross-linking reactions (8Jing S.Q. Tapley P. Barbacid M. Neuron. 1992; 9: 1067-1079Abstract Full Text PDF PubMed Scopus (391) Google Scholar). However, immunoprecipitation experiments using 125I cross-linked to neural tissue or cell lines suggest that an association between the Trk A and p75 may take place (9Huber L.J. Chao M.V. J. Neurosci. Res. 1995; 40: 557-563Crossref PubMed Scopus (120) Google Scholar, 10Ross G.M. Shamovsky I.L. Lawrence G. Solc M. Dostaler S.M. Weaver D.F. Riopelle R.J. Eur. J. Neurosci. 1998; 10: 890-898Crossref PubMed Scopus (71) Google Scholar, 11Gargano N. Levi A. Alema S. J. Neurosci. Res. 1997; 50: 1-12Crossref PubMed Scopus (57) Google Scholar). Co-precipitation of endogenous Trk A and p75 in PC12 cells indicates that the receptor complex may form without NGF (12Yano H. Chao M.V. Pharm. Acta Helv. 2000; 74: 253-260Crossref PubMed Scopus (85) Google Scholar). Photobleaching recovery experiments using fluorescently tagged p75 receptors or using monovalent antibody detection of Trk A have also revealed physical clustering of p75 with Trk A mediated by both intracellular and extracellular domains of p75 and Trk A and augmented by Trk A kinase activity (13Wolf 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 (79) Google Scholar). These studies have recently been extended to other Trk family members with the demonstration of a Trk B and p75 interaction by co-immunoprecipitation mediated by intracellular and extracellular domains (14Bibel M. Hoppe E. Barde Y-A. EMBO J. 1999; 186: 16-22Google Scholar). Furthermore, co-expression of p75 with Trk B was found to modulate the ligand specificity of Trk B (14Bibel M. Hoppe E. Barde Y-A. EMBO J. 1999; 186: 16-22Google Scholar). In addition to altering ligand binding, co-expression of p75 with Trk A can also influence Trk A signaling (15Hantzopoulos P.A. Suri C. Glass D.J. Goldfarb M.P. Yancopoulos G.D. Neuron. 1994; 13: 187-207Abstract Full Text PDF PubMed Scopus (261) Google Scholar, 16Barker P.A. Shooter E.M. Neuron. 1994; 13: 203-215Abstract Full Text PDF PubMed Scopus (369) Google Scholar, 17Verdi J.M. Birren S.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 (312) Google Scholar, 18Berg M.M. Sternberg D.W. Hempstead B.L. Chao M.V. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 857-866Google Scholar). The binding of NGF to Trk A receptors activates its cytoplasmic kinase, resulting in the phosphorylation of cytoplasmic tyrosine residues followed by the binding and activation of multiple proteins, including Shc, PLCγ, FRS-2, and SH2B (19Kaplan D.R. Miller F.D. Curr. Opin. Neurobiol. 2000; 10: 381-391Crossref PubMed Scopus (1670) Google Scholar, 20Qian X. Riccio A. Zhang Y. Ginty D.D. Neuron. 1998; 21: 1017-1029Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 21Meakin S.O. MacDonald J.I. Gryz E.A. Kubu C.J. Verdi J.M. J. Biol. Chem. 1999; 274: 9861-9870Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar), which mediate effects such as survival and differentiation. The p75 receptor is a member of the tumor necrosis factor family of receptors and contains a putative cytoplasmic death domain (22Liepinsh E. Ilag L.L. Otting G. Ibanez C.F. EMBO J. 1997; 16: 4999-5005Crossref PubMed Scopus (257) Google Scholar). Although the p75 receptor can modulate Trk activity, and alter the specificity of Trk receptors for neurotrophin ligands (14Bibel M. Hoppe E. Barde Y-A. EMBO J. 1999; 186: 16-22Google Scholar,16Barker P.A. Shooter E.M. Neuron. 1994; 13: 203-215Abstract Full Text PDF PubMed Scopus (369) Google Scholar, 23Maliartchouk S. Saragovi H.U. J. Neurosci. 1997; 17: 6031-6037Crossref PubMed Google Scholar), the p75 receptor can also mediate cell death when expressed independent of Trk receptors (24Casaccia-Bonnefil P. Carter B.D. Dobrowsky R.T. Chao M.V. Nature. 1996; 383: 716-719Crossref PubMed Scopus (719) Google Scholar, 25Frade J.M. Rodriguez-Tebar A. Barde Y-A. Nature. 1996; 383: 166-168Crossref PubMed Scopus (668) Google Scholar, 26Bamji S.X. Majdan M. Pozniak C.D. Belliveau D.J. Aloyz R. Kohn J.M. Causing C.G. Miller F.D. J. Cell Biol. 1998; 140: 911-923Crossref PubMed Scopus (443) Google Scholar). Neurotrophin binding to p75 can result in sphingomyelinase activation (27Dobrowsky R.T. Carter B.D. Ann. N. Y. Acad. Sci. 1998; 845: 32-45Crossref PubMed Scopus (54) Google Scholar) and recruitment of TRAF-6, a mediator of tumor necrosis factor receptor activation (28Khursigara G. Orlinick J.R. Chao M.V. J. Biol. Chem. 1999; 274: 2597-2600Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar), RhoA (29Yamashita T. Tucker K.L. Barde Y.A. Neuron. 1999; 24: 585-593Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar), and NRIF (30Casademunt E. Carter B.D. Benzel J. Frade J.M. Dechant G. Barde Y.A. EMBO J. 1999; 18: 6050-6061Crossref PubMed Scopus (156) Google Scholar). Thus, receptor-mediated signal transduction by the neurotrophins is unique among polypeptide growth factors because two different transmembrane signaling receptors can be activated by a neurotrophin ligand with distinctive biological outcomes. In addition, the ability of each receptor subunit to modulate the signaling cascades initiated by the co-receptor suggests that the receptors directly interact. One potential mechanism for the p75 receptor's ability to influence high affinity binding may be to increase the effective concentration of neurotrophin at the cell surface and thus enhance NGF binding to Trk A (16Barker P.A. Shooter E.M. Neuron. 1994; 13: 203-215Abstract Full Text PDF PubMed Scopus (369) Google Scholar). A similar mechanism involving heparin sulfate facilitation of fibroblast growth factor binding to FGFR2 has been postulated (31Spivak-Kroizman T. Lemmon M.A. Dikic I. Ladbury J.E. Pinchasi D. Huang J. Jayne M. Crumley G. Schlessinger J. Lax I. Cell. 1994; 79: 1015-1024Abstract Full Text PDF PubMed Scopus (596) Google Scholar), but a more complex role in inducing conformational changes in the FGFR2 complex has become apparent with crystallographic analysis (32Pellegrini L. Burke D.F. von Delft F. Mulloy B. Blundell T.L. Nature. 2000; 407: 1029-1034Crossref PubMed Scopus (630) Google Scholar). Another mechanism for p75 regulation of high affinity site formation is that the conformation of Trk A may be altered in the presence of p75, and this allosteric regulation facilitates Trk A-ligand binding and subsequent signaling functions (7Mahadeo D. Kaplan L. Chao M.V. Hempstead B.L. J. Biol. Chem. 1994; 269: 6884-6891Abstract Full Text PDF PubMed Google Scholar, 10Ross G.M. Shamovsky I.L. Lawrence G. Solc M. Dostaler S.M. Weaver D.F. Riopelle R.J. Eur. J. Neurosci. 1998; 10: 890-898Crossref PubMed Scopus (71) Google Scholar). To test these hypotheses, we undertook structure/function analysis of the p75 and Trk A receptors to identify the domains of each receptor required for high affinity ligand binding. The generation of the baculovirus constructs containing cDNAs for wild type and mutant p75 and Trk A have been described (33Stephens R.M. Loeb D.M. Copeland T.D. Pawson T. Greene L.A. Kaplan D.R. Neuron. 1994; 12: 691-705Abstract Full Text PDF PubMed Scopus (471) Google Scholar), as has the generation of chimeric cDNAs encoding Trk A-torso constructs (34Ross 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 (71) Google Scholar). The cDNAs encoding the epidermal growth factor receptor-p75 chimeras (constructs EN10 and EN31) (35Yan H. Schlessinger J. Chao M.V. Science. 1991; 252: 561-563Crossref PubMed Scopus (95) Google Scholar) were released by EcoRI digestion from the Bluescript vector and ligated into the Nco to EcoRI site of the baculovirus expression vector using polymerase chain reaction primers. The cDNA for the mutant p75 construct (p75–105) (36Yan H. Chao M.V. J. Biol. Chem. 1991; 266: 12099-12104Abstract Full Text PDF PubMed Google Scholar) was excised from Bluescript usingEcoRI digestion and subcloned into the pCMV expression vector. The construction of pCMV5 plasmids encoding the native Trk A and p75 and a Trk A-Trk B chimeric receptor (3.2B) has been described (37Perez P. Coll P.M. Hempstead B.L. Martin-Zanca D. Chao M.V. Mol. Cell. Neurosci. 1995; 6: 97-105Crossref PubMed Scopus (89) Google Scholar). This 3.2B Trk A-Trk B chimera contains the Trk A sequence in the IgGC1 and C2 domains with the leucine-rich repeats, transmembrane, and intracellular domains of Trk B origin. A second Trk A-Trk B chimeric receptor containing the Trk A IgGC1 and C2 domains and the Trk A transmembrane domain with Trk B leucine-rich repeats and intracellular domain was constructed by domain swapping using the polymerase chain reaction primers and techniques described in detail (37Perez P. Coll P.M. Hempstead B.L. Martin-Zanca D. Chao M.V. Mol. Cell. Neurosci. 1995; 6: 97-105Crossref PubMed Scopus (89) Google Scholar). The nucleotide sequence of each cloned fragment from Trk A and Trk B was determined using the dideoxy chain termination method. Sf9 insect cells were maintained in TMN-FH medium from JRH Biosciences supplemented with 9% heat-inactivated fetal bovine serum and 50 µg/ml gentamycin at 28 °C. PC12 nnr5 cells expressing mutant Trk A receptors were cultured as described (33Stephens R.M. Loeb D.M. Copeland T.D. Pawson T. Greene L.A. Kaplan D.R. Neuron. 1994; 12: 691-705Abstract Full Text PDF PubMed Scopus (471) Google Scholar). Human embryonic kidney cell line 293 was maintained as described (35Yan H. Schlessinger J. Chao M.V. Science. 1991; 252: 561-563Crossref PubMed Scopus (95) Google Scholar). To express a single NGF receptor, recombinant baculovirus was added to Sf9 cells (2 − 106 cells in a 25 cm2 flask) at a multiplicity of infection of 1 for the p75 virus or 4 for the Trk A virus. For co-expression experiments, baculovirus encoding Trk A was added to SF9 cells at a m.o.i. of 16 followed immediately by the addition of virus encoding p75 at a m.o.i. of 1. To vary the ratio of infection, the m.o.i. of virus encoding Trk A was varied from 2 to 40, and the m.o.i. of virus encoding p75 was varied from 1 to 10. After 60 h of infection, cells were pelleted by centrifugation, washed in phosphate-buffered saline, and snap frozen in liquid nitrogen. 293 cells were cotransfected with the pCMV p75 or pCMV mutant p75 (p75–105) construct and the pMEX construct, which encodes a neomycin resistance gene, using the calcium phosphate precipitation method (37Perez P. Coll P.M. Hempstead B.L. Martin-Zanca D. Chao M.V. Mol. Cell. Neurosci. 1995; 6: 97-105Crossref PubMed Scopus (89) Google Scholar). Following selection in media containing 250 µg of G418, colonies were subcloned and expanded. The level of expression of native or mutant p75 was determined using whole cell lysates in Western blot analysis with the 9992 antisera specific for the intracellular domain of human p75 (36Yan H. Chao M.V. J. Biol. Chem. 1991; 266: 12099-12104Abstract Full Text PDF PubMed Google Scholar). Clones expressing native or mutant p75 were subsequently transiently transfected with pCMV5 encoding Trk A using the calcium phosphate method and incubation of the cells with DNA for 4 h followed by replacement with fresh media. Twenty eight h after the addition of DNA, cells were harvested in phosphate-buffered saline containing 1 mm EDTA. For membrane binding assays, cells were pelleted and snap frozen in liquid nitrogen. For binding analysis using intact cells, cell suspensions were utilized immediately upon harvesting. Following lysis of cells in radioimmunoprecipitation assay, protein content was determined by the Bio-Rad assay using bovine serum albumin as a standard (38Hempstead B.L. Patil N. Thiel B. Chao M.V. J. Biol. Chem. 1990; 265: 9595-9598Abstract Full Text PDF PubMed Google Scholar). Equivalent concentrations of protein were subjected to separation by SDS-PAGE, blotted onto nitrocellulose, and probed with the 9992 antisera to intracellular epitopes of p75 (7Mahadeo D. Kaplan L. Chao M.V. Hempstead B.L. J. Biol. Chem. 1994; 269: 6884-6891Abstract Full Text PDF PubMed Google Scholar), anti-Trk antisera 203 detecting intracellular epitopes (3Hempstead B.L. Martin-Zanca D. Kaplan D.R. Parada L.F. Chao M.V. Nature. 1991; 350: 678-683Crossref PubMed Scopus (1022) Google Scholar), Trk A-Out detecting extracellular epitopes (39Donovan M.J. Hempstead B. Huber L.J. Kaplan D. Tsoulfas P. Chao M. Parada L. Schofield D. Am. J. Pathol. 1994; 145: 792-801PubMed Google Scholar), or antisera to torso (34Ross 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 (71) Google Scholar) as described. Immunocomplexes were detected by enhanced chemiluminescence technique (Amersham Pharmacia Biotech) and subjected to densitometry. Mouse NGF (Bioproducts for Science, renin free) was radioiodinated using lactoperoxidase and hydrogen peroxide as described (38Hempstead B.L. Patil N. Thiel B. Chao M.V. J. Biol. Chem. 1990; 265: 9595-9598Abstract Full Text PDF PubMed Google Scholar). The specific activity averaged 3000 dpm/fmol and was used within 2 weeks of radioiodination. Crude membrane preparations from snap frozen cells were prepared as described (7Mahadeo D. Kaplan L. Chao M.V. Hempstead B.L. J. Biol. Chem. 1994; 269: 6884-6891Abstract Full Text PDF PubMed Google Scholar). The equilibrium binding assay conditions utilizing membrane preparations have been described in detail (7Mahadeo D. Kaplan L. Chao M.V. Hempstead B.L. J. Biol. Chem. 1994; 269: 6884-6891Abstract Full Text PDF PubMed Google Scholar, 38Hempstead B.L. Patil N. Thiel B. Chao M.V. J. Biol. Chem. 1990; 265: 9595-9598Abstract Full Text PDF PubMed Google Scholar), utilizing 125I-NGF concentrations from 0.0005 to 4 nm, and each condition was assessed in triplicate in the presence or absence of excess unlabeled (0.8 µm) NGF. The specific counts averaged 60–85% of the total counts. In indicated experiments, equilibrium binding studies were performed using the whole cells binding assay (7Mahadeo D. Kaplan L. Chao M.V. Hempstead B.L. J. Biol. Chem. 1994; 269: 6884-6891Abstract Full Text PDF PubMed Google Scholar). Cells were resuspended at 0.75 − 106/ml final concentration, and binding to radioiodinated NGF (0.0005 to 4 nm) in the presence or absence of excess unlabeled NGF (0.8 µm) proceeded for 2 h at 0.4 °C. Cell-bound NGF was separated from free NGF by pelleting through calf serum. The Scatchard plot analysis was performed using the LIGAND program as described (7Mahadeo D. Kaplan L. Chao M.V. Hempstead B.L. J. Biol. Chem. 1994; 269: 6884-6891Abstract Full Text PDF PubMed Google Scholar) and additionally analyzed using the PRISM program to perform nonlinear regression and directly compare curve fits to determine whether they were similar with 95% confidence limits. The level of expression of the p75 and Trk A receptors are highly regulated during neuronal development and in response to injury. To determine the optimal levels of expression of each receptor that confers the maximal percentage of high affinity binding sites, binding studies were performed using cells that expressed varying ratios of Trk A:p75. In prior studies using PC12 cells, we had noted that expression of Trk A:p75 at ratios of 1.0:0.8 yielded a higher percentage of high affinity sites as compared with PC12 cells with a 1:20 ratio of Trk A:p75 (42Hempstead B.L. Rabin S.J. Kaplan L. Reid S. Parada L.F. Kaplan D.R. Neuron. 1992; 9: 883-896Abstract Full Text PDF PubMed Scopus (286) Google Scholar). Transient expression of Trk A and p75 in Sf9 cells using baculovirus expression vectors allowed the two receptors to be expressed at defined ratios by altering the ratio of infectious virus and generated Sf9 cells that expressed high levels of receptors necessary for equilibrium binding studies. The level of expression of each receptor in infected Sf9 cells was determined by Western blot analysis (Fig.1a) in comparison with detergent extracts of Trk A overexpressing PC12 (Trk A-PC12) cells, which express 70,000 Trk A and 90,000 p75 receptors per cell as assessed by kinetic analysis of NGF binding sites in whole cell preparations (7Mahadeo D. Kaplan L. Chao M.V. Hempstead B.L. J. Biol. Chem. 1994; 269: 6884-6891Abstract Full Text PDF PubMed Google Scholar) (Fig. 1 b). Expression of the Trk A receptor at levels significantly in excess of p75 results in predominantly low affinity binding sites. At ratios of Trk A:p75 of ∼20:1, only low affinity binding sites were detected, and these sites exhibited a K d of 1.1 × 10−9m (Fig. 1 e and TablesIand II). Conversely, when p75 was expressed at levels significantly in excess of Trk A (ratio of Trk A:p75 of 1:15), only 3% of the sites exhibited high affinity binding (K d = 1.6 × 10−11m) (Fig. 1 b and Tables I andII). However, as the ratio of receptors became closer to equivalency (ratios of Trk:p75 of 1:5 or 1:0.8), the percentage of high affinity sites (K d = 2.4–2.8 × 10−11m) increased to 7 or 10% of the total binding sites, respectively (Fig. 1, C andD, and Tables I and II). Indeed, the percentage of high affinity sites and the K d of the high affinity site observed in Sf9 cells expressing Trk A:p75 at a 1:0.8 ratio is comparable with that observed using Trk A-overexpressing PC12 cells at a 0.8:1 ratio (7Mahadeo D. Kaplan L. Chao M.V. Hempstead B.L. J. Biol. Chem. 1994; 269: 6884-6891Abstract Full Text PDF PubMed Google Scholar, 42Hempstead B.L. Rabin S.J. Kaplan L. Reid S. Parada L.F. Kaplan D.R. Neuron. 1992; 9: 883-896Abstract Full Text PDF PubMed Scopus (286) Google Scholar), demonstrating that binding results obtained with whole cells and membrane preparations are consistent and that receptor expression in different cell types yields binding sites with similar affinity (3Hempstead B.L. Martin-Zanca D. Kaplan D.R. Parada L.F. Chao M.V. Nature. 1991; 350: 678-683Crossref PubMed Scopus (1022) Google Scholar, 7Mahadeo D. Kaplan L. Chao M.V. Hempstead B.L. J. Biol. Chem. 1994; 269: 6884-6891Abstract Full Text PDF PubMed Google Scholar, 42Hempstead B.L. Rabin S.J. Kaplan L. Reid S. Parada L.F. Kaplan D.R. Neuron. 1992; 9: 883-896Abstract Full Text PDF PubMed Scopus (286) Google Scholar). Thus, the ratio of expression of Trk A to p75 can significantly alter the number of high affinity sites with a higher percentage of the total receptors exhibiting high affinity binding when the receptors are expressed in near equimolar ratios.Table ISchematic representation of the structural domains of the p75 and Trk A receptor required for high affinity NGF bindingTable IIBinding constants and quantitation of binding sites obtained from equilibrium binding sitesExperimental conditionAnalysis of equilibrium bindingLow affinity siteCell typeHigh affinity siteKd (10−11m)BmaxKd (10−9m)BmaxATrkA:p751:15**(2)Sf91.6151-aBmax recorded in fmol/µg.1.24501-aBmax recorded in fmol/µg.1:5 (2)Sf92.4231-aBmax recorded in fmol/µg.1.13001-aBmax recorded in fmol/µg.1:0.8*(2)Sf92.8331-aBmax recorded in fmol/µg.1.33001-aBmax recorded in fmol/µg.20:1**(3)Sf9––1.19901-aBmax recorded in fmol/µg.BTrk + p75*(3)Sf91.71501-aBmax recorded in fmol/µg.0.910101-aBmax recorded in fmol/µg.305 + p75**(3)Sf91.81401-aBmax recorded in fmol/µg.0.834101-aBmax recorded in fmol/µg.303 + p75**(3)Sf97.01401-aBmax recorded in fmol/µg.1.919501-aBmax recorded in fmol/µg.325 + p75**(2)Sf94.0901.023401-aBmax recorded in fmol/µg.CTrk + p75*(2)2932.3220.81601-bBmax recorded in fmol/106 cells.3.3 + p75**(3)2932.6111-bBmax recorded in fmol/106 cells.1.0601-bBmax recorded in fmol/106 cells.3.2 + p75**(3)29318.2401-bBmax recorded in fmol/106 cells.0.91501-bBmax recorded in fmol/106 cells.K538 + p75**(4)NC––0.93001-aBmax recorded in fmol/µg.DTrk A + EGF-p75(10)*(3)Sf91.9241-aBmax recorded in fmol/µg.1.31401-aBmax recorded in fmol/µg.Trk A + EGFR-p75(31)**(3)Sf9––1.62001-aBmax recorded in fmol/µg.Trk A + p75(50)(2)2932.8161-bBmax recorded in fmol/106 cells.1.41801-bBmax recorded in fmol/106 cells.The PRISM program was used for linear regression analysis of the equilibrium binding data, and Scatchard transformation was performed to generate the K d and B max of each site. Curve fits obtained by nonlinear regression analysis were compared to determine whether they were similar within 95% confidence limits. The number of experiments analyzed per condition were analyzed, and conditions that were different (p < 0.05) from the input curve (*) are denoted (**). (A) See Fig. 1; (B) See Fig. 2; (C) See Figs. 3 and 4; and (D) See Figs. 5 and 6.1-a Bmax recorded in fmol/µg.1-b Bmax recorded in fmol/106 cells. Open table in a new tab The PRISM program was used for linear regression analysis of the equilibrium binding data, and Scatchard transformation was performed to generate the K d and B max of each site. Curve fits obtained by nonlinear regression analysis were compared to determine whether they were similar within 95% confidence limits. The number of experiments analyzed per condition were analyzed, and conditions that were different (p < 0.05) from the input curve (*) are denoted (**). (A) See Fig. 1; (B) See Fig. 2; (C) See Figs. 3 and 4; and (D) See Figs. 5 and 6. The region of the extracellular domain of Trk A that mediates binding to NGF has been mapped to the juxtamembrane IgG C2 domains (40Urfer R. Tsoulfas P. Soppet D. Escandon E. Parada L.F. Presta L.G. EMBO J. 1994; 13: 5896-5909Crossref PubMed Scopus (101) Google Scholar, 41Ryden M. Murray-Rust J. Glass D. Ilag L.M. Yancopoulos G.D. McDonald N.Q. Ibanez C.F. EMBO J. 1995; 14: 1979-1990Crossref PubMed Scopus (113) Google Scholar). However, the effects of alterations within the transmembrane and cytoplasmic domains upon NGF binding are unknown. To assess the role of each of these domains, the transmembrane and cytoplasmic domains of the torso receptor kinase were exchanged with the Trk A receptor. Torso is a distantly related receptor-tyrosine kinase to Trk A, and a series of chimeric Trk A-torso receptors were generated (34Ross 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 (71) Google Scholar) to test the" @default.
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• W2089124684 title "The Cytoplasmic and Transmembrane Domains of the p75 and Trk A Receptors Regulate High Affinity Binding to Nerve Growth Factor" @default.
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