APPL1 and GIPC1 share similar codistribution patterns in sympathetic neurons. Endogenous APPL1 (A) and GIPC1 (B) display punctate patterns of distribution in axons of sympathetic neurons cultured in medium containing NGF. Neurons from newborn rat SCG were immunostained with an antibody against APPL1 or GIPC1 as indicated. (C) As a negative control, primary antibody was omitted. (D) Anti-Flag staining (red) of an axon from a sympathetic neuron transiently transfected to express Flag-GIPC1. (E) Distribution of endogenous APPL1 (green) as determined by immunofluorescence staining with anti-APPL1 antibody. The same field is shown in panel D. Arrowheads mark examples of Flag-GIPC1 and endogenous APPL1 codistribution (D and E). (F) Merged image of panels D and E. Bar, 10 μm.

Identification of APPL1 as a binding partner for GIPC1. Cellular proteins precipitated with a GST fusion of full-length GIPC1 were isolated from adult rat brain extracts. Interacting proteins were resolved by SDS-PAGE and detected by staining with Coomassie brilliant blue. Total cellular proteins representing the starting material (SM) are shown in the first lane. Addition of GST alone to lysate and incubation of GST-GIPC1 in the absence of lysate were included as negative controls. The GST-GIPC1 band is marked with an arrow. Bands specifically precipitated with GST-GIPC1 (marked with arrowheads) were excised from the gel and digested with trypsin. The resulting peptides were sequenced by mass spectrometry.

GIPC1 and APPL1 associate in cells. (A) APPL1, TrkA, and pTrkA coprecipitate with GIPC1 in PC12 (6-24) cells. Endogenous GIPC1 was precipitated from lysates of cells treated with 50 ng/ml NGF for 10 min (+) or from untreated cells (−). The presence of endogenous APPL1, TrkA, and pTrkA in the precipitates was detected by immunoblotting (IB) with anti-APPL1, anti-TrkA, and antiphosphotyrosine antibodies, respectively. Immunoprecipitation with preimmune serum (Pre-Imm IP) was included as a negative control. TrkA, pTrkA, APPL1, and GIPC1 proteins are marked with arrowheads. The upper band in the anti-GIPC1 immunoblot is the immunoglobulin heavy chain. (B) APPL1 coprecipitates with GIPC1 in rat primary sympathetic neurons. Endogenous GIPC1 was precipitated from sympathetic neuron lysates treated with 50 ng/ml NGF for 10 min. Immunoblots were probed with the indicated antibodies.

pTrkA, APPL1, and GIPC1 are present together in endosomal subfractions. (A) Fractions of endosomes from NGF-treated PC12 cells were isolated and then separated on the basis of size by velocity centrifugation. Five velocity gradient fractions were collected, and each pool was further resolved on flotation equilibrium gradients. Data from an equilibrium gradient of velocity gradient fraction 2 are shown. Equilibrium gradient fractions were collected such that endosomes of higher density are contained in lower-numbered fractions. Proteins were resolved by SDS-PAGE, and the immunoblot (IB) was probed with the indicated antibodies. (B) Quantification and graphical representation of the data shown in panel A. The density of each fraction was determined by refractive index measurements. Chemiluminescent signal strength from each of the pTrk, APPL1, and GIPC1 bands was quantified and plotted against density. Data shown in panels A and B are representative of three separate experiments.

Reduction of APPL1 and GIPC1 protein levels by RNA interference decreases ERK and Akt activation but does not affect TrkA tyrosine phosphorylation. (A) pERK and pAkt are reduced in APPL1 or GIPC1 siRNA-treated cells. PC12 (6-24) cells were transfected with a negative-control siRNA or siRNAs targeting GIPC1 or APPL1. After NGF withdrawal, cells were treated with 50 ng/ml NGF for 10 min (+NGF) or left untreated (−NGF). Equal amounts of cell extract (30 μg total protein) were analyzed by immunoblotting (IB) with the indicated antibodies. Levels of ERK1 and ERK2 were detected as a control for the specificity of the siRNA treatment. (B) MEK activation is decreased whereas ShcA phosphorylation is unaffected in APPL1 and GIPC1 siRNA-treated cells. The same lysates from the experiments described for panel A were probed with antibodies against pMEK, MEK, pShcA (pY317), ShcA, and Akt. (C) Neither TrkA levels nor levels of TrkA total tyrosine phosphorylation are affected by reduction of APPL1 and GIPC1 protein levels. TrkA was immunoprecipitated (IP) from 100 μg of the lysates from the same experiment shown in panels A and B. Immunoblots were probed with antibodies against TrkA and phosphotyrosine.

Reduction of APPL1 or GIPC1 levels decreases NGF-induced neurite outgrowth. (A) GFP epifluorescence in fields of NGF-differentiated PC12 (6-24) cells following siRNA treatment. Cells were cotransfected with the indicated siRNA and with pEGFP to allow for visualization of neurites. Twenty-four hours following transfection, cells were differentiated by treatment with 50 ng/ml NGF for 48 h. Bar, 10 μm. (B) Effect of RNA interference-mediated reduction of APPL1 or GIPC1 levels on neurite outgrowth in PC12 (6-24) cells. Neurite lengths were measured 48 h following NGF treatment, 72 h posttransfection. Mean neurite lengths per cell from three independent experiments (n = 3) are shown, along with standard errors. Cells treated with siRNAs targeting APPL1 or GIPC1 have significantly shorter average neurite lengths than those transfected with the control siRNA (P < 0.02, Bonferroni and Tukey tests on a paired design). (C) Levels of APPL1 and GIPC1 protein are reduced in the cells shown in panel A. Following immunofluorescence analysis, cells were lysed and levels of APPL1 and GIPC1 were analyzed by immunoblotting (IB) with the indicated antibodies. ERK levels provided a control for equal loading.

The PDZ domain of GIPC1 binds directly to the C terminus of APPL1. (A) Schematic representations of the domain architecture of GIPC1 (left) and APPL1 (right). GST, Flag, and His epitope-tagged fusions of fragments and mutants of GIPC1 and APPL1 are shown. (B) GST fusions of full-length GIPC1 or fragments of GIPC1 containing the PDZ domain specifically precipitate YFP-APPL1 from lysates of transfected COS7 cells. Cell lysates were incubated with the GST fusion proteins as indicated, and the resulting complexes were analyzed by immunoblotting (IB) with anti-GFP antibody. (C) The PDZ domain of GIPC1 is necessary and sufficient for direct association with the C-terminal region (C-term) of APPL1. Purified fragments of APPL1 fused to a His epitope were incubated with purified, Sepharose-immobilized GST fusions of the GIPC1 N terminus (N-term), GIPC1 N terminus plus PDZ (N-term + PDZ), or GST alone. Anti-His antibody was used to detect His-APPL1 proteins by immunoblotting. (D) Removal of the C-terminal alanine residue of APPL1 (ΔC-term Ala) abrogates its association with GIPC1. COS7 cells were transiently transfected to express either wild-type Flag-APPL1 or Flag-APPL1 ΔC-term Ala. Cell lysates were incubated with GST fusion proteins as indicated and analyzed by immunoblotting with anti-Flag antibody. Lysates of transfected cells show expression levels of the wild-type and truncated proteins. (E) Mutation of the carboxylate-binding loop within the GIPC1 PDZ domain eliminates its interaction with APPL1. Flag-GIPC1 and the Flag-GIPC1 PDZ mutant were immunoprecipitated (IP) from transfected COS7 cells with anti-Flag antibody. Endogenous APPL1 associated with Flag-GIPC1 was detected with antibodies against APPL1 (top). Levels of Flag-GIPC1 proteins were determined using the anti-Flag antibody (bottom).

Endogenous APPL1 coprecipitates with TrkA. (A) Sympathetic neurons infected with Flag-TrkA adenovirus were subjected to NGF withdrawal and then treated with 50 ng/ml NGF for 10 min (+NGF) or left untreated (−NGF). TrkA was immunoprecipitated (IP) from lysates by use of an anti-pan-Trk antibody. Precipitation of lysates with protein A Sepharose (Prot A) alone and the incubation of pan-Trk antibody in the absence of lysate were included as negative controls. Immunoblots (IB) were probed with the indicated antibodies. APPL1, pTrkA, and TrkA bands are marked with arrowheads. The additional band in the TrkA immunoblot corresponds to an immature, underglycosylated form of TrkA. (B) Association of endogenous APPL1 with TrkA does not require TrkA catalytic activity. Experiments were performed as described for panel A, but cells were treated either with 200 nM of the inhibitor K252a in DMSO (+) or with DMSO alone (−). (C) TrkA and APPL1 display partially overlapping distributions in the axons of rat primary sympathetic neurons cultured in medium containing NGF. Confocal images were taken of neurons stained with antibodies against APPL1 (green) or TrkA (red). The rightmost panel shows the merged image. Arrowheads mark examples of the overlap between TrkA and APPL1 distribution. Bar, 5 μm.

The PTB domain of APPL1 binds directly to Trk receptors in a phosphotyrosine-independent manner. (A) APPL1 binds to TrkA peptides corresponding to the Shc-binding site. Biotinylated phosphopeptide containing the TrkA sequence PHIIENPQpYFSD or the unphosphorylated version of the peptide was bound to streptavidin beads and incubated with lysates of PC12 (6-24) cells. Streptavidin beads bound only to biotin were included as a negative control. Endogenous APPL1 was detected by immunoblotting (IB) with anti-APPL1 antibodies, and blots were also probed with anti-ShcA antibodies as a positive control. (B) pTrkA associates with the PTB domain of APPL1. NGF was withdrawn from primary cultures of sympathetic neurons, and the cells were treated with 50 ng/ml NGF for 10 min (+) or left untreated (−). Lysates were incubated with GST-APPL1 PTB or GST alone as a negative control or were immunoprecipitated (IP) with a pan-Trk antibody as a positive control. Endogenous pTrkA representing the NGF-activated form of TrkA was detected by immunoblotting with antiphosphotyrosine antibody. (C) The association between the APPL1 PTB domain and TrkA is phosphotyrosine independent. Experiments were performed as described for panel B. Immunoblots were probed with anti-TrkA antibody to detect both phosphorylated and unphosphorylated forms of TrkA. The mature, glycosylated TrkA protein is marked with an arrowhead. The additional lower band in the TrkA immunoblot corresponds to an immature, underglycosylated form of TrkA. (D) The PTB domain of APPL1 interacts directly with the juxtamembrane region of TrkA. A purified His-tagged fragment of TrkA containing the juxtamembrane region was incubated with GST fusions of the APPL1 BAR, PH, and PTB domains or with GST alone. Purified His-TrkA juxtamembrane protein (50 ng) is included as a control. The immunoblot was probed with antibody against the His tag.