Article Figures & Data
Figures
- Fig. 1.
Dominant inhibitory effect of naturally occurring truncated trkB receptors in 45Ca efflux assay. Oocytes were injected with cRNA (2 ng/oocyte) and were then loaded with45Ca as described in Materials and Methods. After45Ca efflux levels stabilized, 250 ng/ml BDNF was added to the medium (indicated by arrow). Mean values from four determinations ± SD are shown. A, Comparison of BDNF-induced 45Ca efflux in oocytes expressing the full-length trkB receptor (closed squares) trkB.T1 (closed circles), trkB.T2 (closed triangles), or water control (open triangles). B, Dominant inhibitory effect of trkB.T1. Oocytes were injected with trkB cRNA (2 ng/oocyte) + varying quantities of trkB.T1 (0, 2, 8, or 18 ng/oocyte).C, Dominant inhibitory effect of trkB.T2. Oocytes were injected with 2 ng/oocyte trkB cRNA + varying quantities of trkB.T2 (0, 2, 8, or 18 ng/oocyte). D, Dominant inhibitory effect of trkB.T1 and trkB.T2 correlates with loss of trkB homodimers. A binomial model of dimer association (see Results) was used to predict the number of trkB homodimers (open squares). Peak45Ca efflux values for trkB.T1-injected (closed circles) and trkB.T2-injected (closed triangles) oocytes are plotted as a function of truncated:full-length trkB cRNA.
- Fig. 2.
ATP-binding mutant of trkB inhibits BDNF signaling at the level of receptor tyrosine phosphorylation. A, ATP-binding site mutation abolishes BDNF-induced45Ca efflux response. Oocytes were injected with cRNA (2 ng/oocyte) encoding the full-length trkB receptor (closed squares) or its ATP-binding mutant. After45Ca efflux had stabilized, BDNF was added to the medium (250 ng/ml, arrow). B, Dominant inhibitory effect of the ATP-binding mutant. Oocytes were injected with 2 ng/oocyte trkB cRNA + varying quantities of ATP-binding mutant (0, 2, 8, and 18 ng/oocyte). C, Decrease in maximal45Ca efflux responses and trkB tyrosine phosphorylation correlate with a loss of trkB homodimers. A binomial model (see Results) was used to predict the number of trkB homodimers (open squares). Percent maximum trkB tyrosine phosphorylation was determined by labeling trkB ± ATP-binding mutant-expressing oocytes with 35S, stimulating with BDNF, immunoprecipitating lysates with APT antibody, separating on SDS-PAGE gels, and then quantitating bands by PhosphorImager analysis (see Materials and Methods). Data are displayed graphically as percent maximum tyrosine phosphorylation (closed circles). Finally, peak 45Ca efflux responses for ATP-binding mutant-expressing oocytes are plotted as a function of the ratio of truncated:full-length trkB cRNA (closed squares).
- Fig. 4.
Epitope tags distinguish the ATP-binding mutant from the wild-type trkB receptor. A, COS cells transfected with either vector control (lane 1) or trkBIVH (lane 2). Lysates were immunoprecipitated with IVH Ab (12CA5), separated by 6% SDS-PAGE, then immunoblotted with IVH Ab. B, COS cells transfected with either vector control (lane 1) or ATPmutmyc (lane 2). Lysates were immunoprecipitated with anti-myc Ab (9E10), separated by 6% SDS-PAGE, and then blotted with anti-myc Abs.
- Fig. 5.
Expression levels of wild-type and ATP-binding mutant reflect quantities of cRNA injected. A, IVH ECL blot of oocytes injected with trkBIVH ± ATPmutmyc. Oocytes were injected as described in Materials and Methods, lysed 36 hr later with RIPA buffer, separated on 7% SDS-PAGE gels, and then immunoblotted with IVH Ab (12CA5). Oocytes were injected with sterile water (lane 1), trkBIVH (1 ng; lane 2), or ATPMutmyc + trkBIVH at a ratio of 4:1 (4 ng/1 ng/oocyte; lane 3) or 9:1 (9 ng/1 ng/oocyte;lane 4). Lysates collected from 6 oocytes were loaded per lane. B, Myc ECL blot of oocytes co-injected with trkBIVH ± ATPmutmyc. Oocytes were injected and lysed as described above. Lysates from 2 oocytes were loaded per lane. Proteins were separated on 7% SDS-PAGE gels and then immunoblotted with myc Ab (9E10). Oocytes were injected with trkBIVH only (1 ng/oocyte; lane 1) or ATPMutmyc:trkBIVH at a ratio of 9:1 (9 ng/1 ng/oocyte; lane 2) or 4:1 (4 ng/1 ng/oocyte; lane 3).
- Fig. 3.
Intermolecular phosphorylation of ATP-binding mutant by wild-type gp145trkB.A, Schematic diagram of epitope-tagged wild-type and mutant trkB receptors. PCR was used to attach an IVH tag to the C terminus of the wild-type trkB receptor (trkBIVH) and myc tag to the C terminus of the ATP-binding mutant (ATPMutmyc) as described in Materials and Methods. Amino acid numbers are listed for the extracellular (EC) or transmembrane domains (TM), ATP-binding site (Lys or Met), and epitope tags (IVH or Myc). B, BDNF induces rapid tyrosine phosphorylation of trkBIVH receptors. COS cells were transfected with vector control (lanes 1, 2) or trkBIVH (lanes 3, 4). Cells in lanes 2 and 4 were stimulated for 5 min with 100 ng/ml BDNF. Lysates were immunoprecipitated with IVH Ab, separated by 6% SDS-PAGE, and then immunoblotted with the APT Ab 4G10. C, Intermolecular tyrosine phosphorylation of ATPMutmyc by trkBIVH. COS cells were transfected with vector alone (lanes 1, 6), ATPMutmyc (lane 2), ATPmutmyc + trkBIVH (lanes 3, 4), or trkBIVH alone (lane 5). Cells in lane 4 were pretreated with 50 mm sodium orthovanadate for 3 hr before lysis. Cells were stimulated with BDNF, then immunoprecipitated with either anti-myc (lanes 1–4) or anti-IVH Abs (lanes 5, 6). Proteins were separated by 6% SDS-PAGE and were then immunoblotted with APT Ab. The location of gp145trkB is indicated by anarrow.
