Sorting by the cytoplasmic domain of the amyloid precursor protein binding receptor SorLA

Abstract

SorLA/LR11 (250 kDa) is the largest and most composite member of the Vps10p-domain receptors, a family of type 1 proteins preferentially expressed in neuronal tissue. SorLA binds several ligands, including neurotensin, platelet-derived growth factor-bb, and lipoprotein lipase, and via complex-formation with the amyloid precursor protein it downregulates generation of Alzheimer's disease-associated Abeta-peptide. The receptor is mainly located in vesicles, suggesting a function in protein sorting and transport. Here we examined SorLA's trafficking using full-length and chimeric receptors and find that its cytoplasmic tail mediates efficient Golgi body-endosome transport, as well as AP-2 complex-dependent endocytosis. Functional sorting sites were mapped to an acidic cluster-dileucine-like motif and to a GGA binding site in the C terminus. Experiments in permanently or transiently AP-1 mu1-chain-deficient cells established that the AP-1 adaptor complex is essential to SorLA's transport between Golgi membranes and endosomes. Our results further implicate the GGA proteins in SorLA trafficking and provide evidence that SNX1 and Vps35, as parts of the retromer complex or possibly in a separate context, are engaged in retraction of the receptor from endosomes.

FIG. 1.

Localization of SorLA, Sortilin, and endogenous LRP in stably transfected 293 cells. (A) Double transfectants were fixed and stained with monoclonal anti-Sortilin (green) and polyclonal anti-SorLA (red) as primary antibodies. (B) Subcellular fractionation of double transfected 293 cells. Fractions 7 to 9 of the velocity gradient (VG) were subjected to equilibrium gradient (EG) centrifugation. Fraction were analyzed by Western blotting with anti-SorLA, anti-Sortilin, and anti-LRP antibodies. The bars show peak fractions of the indicated markers.

FIG. 2.

(A) Primary sequence of the SorLA cd. (B) Schematic presentation of the chimeric receptors and the cd constructs used in the study. The luminal domains of the IL-2R and the MPR300 are indicated. Point mutations and truncations introduced in the SorLA-cd of the chimeras are shown in the lower panel.

FIG. 3.

(A) Time course of ¹²⁵I-labeled anti-Tac internalization in CHO transfectants expressing IL-2R/SorLA chimeras. Internalization was defined as the amount of cell-associated radioactivity not released upon incubation at pH 2.5. Each point represents a mean (± the standard deviation) of three experiments. All values are relative to the amount of releasable tracer at zero time. (B) Analysis of internalization by confocal microscopy. CHO transfectants expressing IL-2R/SorLA chimeras comprising the wt SorLA-cd (upper panels) or a cd with a disrupted AC (lower panels) were incubated with unlabeled anti-Tac (4°C, 2 h), washed, and incubated in warm medium at zero time. At the times indicated, the cells were fixed and internalized anti-Tac was visualized by using Alexa 488-conjugated goat anti-mouse immunoglobulin (green). Golgi body staining (TGN38) is shown in blue. (C) Similar to the experiment in panel B but performed with corresponding full-length SorLA constructs.

FIG. 4.

(A) AP-2 in SorLA internalization. The upper panel shows pull-down of AP-2 α-chain from brain extracts (Western blot). Precipitation was performed with GST or GST-fusion proteins containing the cd of SorLA wt or Δ(30-54) or of SorCS1c. The middle panel shows a Western blot analysis of AP-2 μ-chain in siRNA-treated (□) and untreated (•) 293 cells and the influence of AP-2 knockdown on the internalization of anti-Tac-IL-2R/SorLA complexes in the same cells. The lower panel (confocal microscopy) shows localization of corresponding receptor-antibody complexes (bound at 4°C) after 30 min at 37°C in untreated (left) cells and cells subjected to RNAi (right). (B) Routing of internalized wt SorLA in transfected 293 cells. Unlabeled (a) or conjugated (b and c) anti-SorLA was bound to cells at 4°C prior to incubation in warm medium. At the times indicated, the cells were fixed and analyzed by electron (a) or confocal microscopy (b and c). (a) Receptor-anti-SorLA complexes in coated pit at zero time. Staining was done by using goat anti-rabbit immunoglobulin coupled to gold beads. (b) Complexes (red) accumulating in early endosomes of cells stably expressing GFP-EEA1 (green). The lower panels show the localization of receptor complexes and TGN46 (c) or receptor complexes and Lamp-1 (d) in cells stained by anti-SorLA (red) and anti-TGN46 or anti-Lamp-1(green), followed by conjugated secondary immunoglobulin after fixation. Double-stained vesicles are indicated by arrows.

FIG. 5.

Morphology of late endosomes or lysosomes in mpr^−/− cells before and after transfection with MPR300/SorLA (upper panel). Cells were fixed and stained using anti-Lamp-1 immunoglobulin and Alexa 488 goat anti-rabbit antibody. The lower panel shows the percentage of the given hydrolases that was detected in the culture medium of untransfected mpr^−/− cells and of cells stably transfected with either wt MPR300 or MPR300/SorLA. Values obtained in the absence or presence of M6P are shown. Each value represents a mean (± the standard deviation) of three separate experiments.

FIG. 6.

Sorting and processing of cathepsin D in untransfected mpr^−/− cells and in cells transfected with MPR300/SorLA. (A) The cells were biolabeled in the presence of brefeldin A, washed, and reincubated in full medium. At the times indicated, incubation was stopped and cathepsin D was immunoprecipitated and analyzed by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An autoradiography of a diphenyloxazole-fluorographed gel is shown, and the percentage of labeled protein found in the cell lysate (c) and in the corresponding medium (m) is given. (B) Western blot performed on lysates of transfected and untransfected mpr^−/− cells using anti-cathepsin D immunoglobulin. The cells were cultured in the absence or presence of M6P. The positions of the proforms (p) and immature forms (I) of cathepsin D and of the double-chain (db) and single-chain (sm) mature forms are indicated by arrows.

FIG. 7.

Influence of AP-1 deficiency on SorLA sorting. (A) Full-length SorLA in wt and in AP-1-deficient MEFs. Transfectants were fixed and analyzed by confocal microscopy by using anti-SorLA immunoglobulin and Alexa 488-goat anti-rabbit immunoglobulin. (B) SorLA in subcellular fractions of wt and AP-1-deficient MEFs. Fractions obtained after velocity gradient centrifugation (VG) and additional separation (selected fractions) on an equilibrium gradient (EQ) were analyzed by Western blotting with anti-SorLA antibodies. (C) Pull-down of AP-1 from brain lysates. Precipitations were performed with GST or GST-fusion proteins containing the cd of SorLA [wt or Δ(30-54)] or of SorCS1c and analyzed by Western blotting and anti-AP-1 γ-chain immunoglobulin. (D) Sorting of β-glucuronidase in wt mpr^−/− cells and in cells expressing MPR300/SorLA. Western blots show expression of the AP-1 μ-chain before and after RNAi. The hydrolase activity detected in the culture medium (with [−] or without [+] M6P) is given as a percentage of total activity in medium and cells. (E) Western blot showing different distribution in equilibrium gradient (EQ) fractions of full-length SorLA and a SorLA construct with a C-terminal truncation (ΔM⁵¹VIA⁵⁴) disrupting the GGA binding site. Bars indicate the position of vti1a and vit1b peak fractions.

FIG. 8.

Expression levels of endogenous SorLA and Sortilin in HepG2 cells upon SNX1 (A) and Vps35 (B) knockdown. (A) Untreated cells (lane 1) and cells subjected to SNX1 RNAi (lanes 2 and 3) were cultured for 60 h prior to analysis of cell lysates by Western blotting. Enzyme inhibitors (Leu/Pep) preventing lysosomal degradation of receptors (lane 3) were added 36 h after transfection with siRNA. (B) Similar experiments showing Western blot detection of SorLA and Sortilin in cells exposed to Vps35 RNAi. Actin levels are shown for the control.