Low density lipoprotein receptor-related protein 1 (LRP1) forms a signaling complex with platelet-derived growth factor receptor-beta in endosomes and regulates activation of the MAPK pathway

Abstract

In addition to its endocytic function, the low density lipoprotein receptor-related protein 1 (LRP1) also contributes to cell signaling events. In the current study, the potential of LRP1 to modulate the platelet-derived growth factor (PDGF) signaling pathway was investigated. PDGF is a key regulator of cell migration and proliferation and mediates the tyrosine phosphorylation of LRP1 within its cytoplasmic domain. In WI-38 fibroblasts, PDGF-mediated LRP1 tyrosine phosphorylation occurred at 37 degrees C but not at 4 degrees C, where endocytosis is minimized. Furthermore, blockade of endocytosis with the dynamin inhibitor, dynasore, also prevented PDGF-mediated LRP1 tyrosine phosphorylation. Immunofluorescence studies revealed co-localization of LRP1 with the PDGF receptor after PDGF treatment within endosomal compartments, whereas surface biotinylation experiments confirmed that phosphorylated LRP1 primarily originates from intracellular compartments. Together, the data reveal the association of these two receptors in endosomal compartments where they form a signaling complex. To study the contribution of LRP1 to PDGF signaling, we used mouse embryonic fibroblasts genetically deficient in LRP1 and identified phenotypic changes in these cell lines in response to PDGF stimulation by performing phospho-site profiling. Of 38 phosphorylated proteins analyzed, 8 were significantly different in LRP1 deficient fibroblasts and were restored when LRP1 was expressed back in these cells. Importantly, the results revealed that LRP1 expression is necessary for PDGF-mediated activation of ERK. Overall, the studies reveal that LRP1 associates with the PDGF receptor in endosomal compartments and modulates its signaling properties affecting the MAPK and Akt/phosphatidylinositol 3-kinase pathways.

FIGURE 1.

PDGF-mediated tyrosine phosphorylation of LRP1 preferentially occurs during endocytosis. A, purified recombinant PDGFR-β-kd was incubated with GST (lane 1) or GST-LRP1-ICD (lane 3) in the presence of [γ-³²P]ATP at 37 °C for 10 min. GST (lane 2) and GST-LRP1-ICD (lane 4) were incubated with [γ-³²P]ATP in the absence of PDGFR-β-kd as controls. The incorporation of ³²P into the proteins was assessed by autoradiography after SDS-PAGE. Autophosphorylation of PDGFR-β-kd was detected at 65 kDa. B, WI-38 fibroblasts kept at either 4 or 37 °C were incubated with or without PDGF for 15 min. LRP1 was subjected to immunoprecipitation (IP) and then blotted with anti-phosphotyrosine IgG (top) or anti-LRP1 IgG (bottom). The control panel (left) shows immunoprecipitation with non-immune IgG (NI). C, shown is a time course for PDGF-induced LRP1 tyrosine phosphorylation at 4 °C. WI-38 fibroblasts were incubated for 10 min with PDGF at 37 °C (left) or for the indicated times at 4 °C (right). LRP1 tyrosine phosphorylation was detected after immunoprecipitation with anti-LRP1 IgG using anti-phosphotyrosine IgG. D, WI-38 fibroblasts were chilled to 4 °C and incubated with PDGF-B for 1 h. Cells were washed, and the temperature was raised to 37 °C to allow endocytosis. Levels of phospho-PDGFR (black circles) or phospho-LRP1 (gray circles) were measured by immunoblot analysis. The experiments was performed in duplicate. E, representative immunoblots from which the data in panel D were derived show the time course of PDGFR-β phosphorylation, LRP1 phosphorylation, and phospho-ERK generation.

FIGURE 2.

Blockade of endocytosis prevents PDGF-mediated tyrosine phosphorylation of LRP1. Cells treated with dynamin inhibitor fail to internalize LRP1 ligand RAP (A) and PDGF-B (B). WI-38 cells were serum-starved and incubated with or without dynasore (200 μm) at 37 °C for 1 h. ¹²⁵I-RAP (10 nm) or ¹²⁵I-labeled PDGF (10 ng/ml) were incubated with the cells at 37 °C for 20 min. Radioactivity released from the cell surface or associated with cell pellets was measured. C, WI-38 cells were serum-starved and incubated with or without dynasore (200 μm) at 37 °C for 1 h and were then incubated with or without PDGF-B for 15 min at 37 °C. Cells were lysed and analyzed by immunoprecipitation with anti-LRP1 polyclonal antibody (R2629). Both cell lysates (input) and immunoprecipitates (IP) were separated on 4–12% SDS gels and analyzed by immunoblotting. D, WI-38 cells were chilled to 4 °C for 1 h and either left untreated (lane 1) or treated with PDGF-β for 1 h at 4 °C. PDGF-β-treated cells were either left at 4 °C (lane 2) or shifted to 37 °C for 7 min (lane 3). All cells were incubated with dithiobispropionimidate cross-linker for 2 h at 4 °C and then lysed. Cell lysates and immunoprecipitates were analyzed as in panel C. E, shown are densitometry measurements of PDGFR-β co-immunoprecipitating with LRP1.

FIGURE 3.

Functional LRP1 co-localizes with PDGFR-β after activation by PDGF. WI-38 fibroblasts were incubated with monoclonal antibody 5A6 to label the endocytic pool of LRP1. The cells were then chilled and incubated with PDGF (30 nm) for 1 h at 4 °C before raising the temperature to 37 °C for 12 min. Fixed and permeabilized cells were then stained for PDGFR-β. The position of the nucleus is outlined by dashed lines. The scale bar is 5 μm.

FIGURE 4.

Activated PDGFR-β and LRP1 co-localize in both early and late endosomal compartments. After incubation with PDGF-B for 15 min, WI38 fibroblasts were fixed and stained for LRP1 and PDGF-β as well as for early endosomes using anti-EEA1 and recycling endosomes using anti-Rab11. The position of the nucleus is outlined by dashed lines. The scale bar is 5 μm.

FIGURE 5.

PDGF-mediated tyrosine phosphorylation of LRP1 by the F5 chimeric PDGFR/M-CSF receptor mutant is delayed. NIH 3T3 cells transfected with wild type chimeric PDGF/M-CSF receptor (ChiR(WT)) or the F5 mutant (ChiR(F5)) were serum-starved and then incubated with M-CSF. A, at the indicated times cell extracts were prepared, and levels of phosphorylated ChiR were detected by phosphotyrosine immunoblots after immunoprecipitation of the ChiR. B, LRP1 tyrosine phosphorylation was detected in the extracts after immunoprecipitation with anti-LRP1 IgG. C, phosphotyrosine LRP1 levels were quantified and are plotted for the wild type (WT, closed circles) and F5 mutant receptor (open circles).

FIGURE 6.

Internal pools of LRP1 are phosphorylated upon PDGF treatment. A, WI-38 fibroblasts were chilled to stop endocytosis, and cell surface proteins were biotinylated. Then cells were incubated without or with PDGF-B (40 nm) for 10 min at 37 °C, and LRP1 was immunoprecipitated with anti-LRP IgG. This was followed by Neutravidin-Sepharose precipitation i to separate biotinylated LRP1 from LRP1 that was not biotinylated. The biotin labeled (Surface) and unlabeled (Internal) LRP1 was subjected to PAGE and probed first with anti-phosphotyrosine antibody (top panel) and then with monoclonal anti-LRP1 antibody (bottom panel). B, control experiments confirm that the PDGFR-β was activated during this experiment. Immunoblot of cell extracts for phospho-PDGFR-β, total PDGFR-β, phospho-ERK, and total ERK.

FIGURE 7.

Kinetworks^TM KPSS-1.3 phosphoprotein analysis of LRP1^+/−, LRP1^−/−, and B41 clones after PDGF-induced signaling. A, immunoblot analysis for LRP1 expression in cell extracts from LRP1^−/+, LRP1^−/−, and B41 clones using an antibody against LRP1-α subunit is shown. B–D, cells were serum-starved overnight then stimulated with 50 ng/ml PDGF-β for 15 min at 37 °C. Whole cell lysates were prepared and analyzed by phospho-site profiling where the expression and phosphorylation states of 38 signaling proteins were analyzed. Five phosphoproteins were significantly altered (circled) in MEFs expressing LRP1 (B) when compared with LRP1-deficient cell line (C). These proteins were restored in the B41 clones (D). The phosphoproteins are MEK1/2 (S18+S222) (1), Rb (S780) (2), Jun (S73) (3), Erk1 (T202+Y204) (4), and CDK1/2 (Y15) (5). The experiment was performed in duplicate.

FIGURE 8.

LRP1 expression influences activation of the MAPK pathway. Quantification of the results from Fig. 7 in LRP1^+/−, LRP1^−/−, and B41 clones is shown. Several signaling pathways affected by LRP1 expression are phosphorylation states of MAPK pathway-related proteins (A) and cell cycle-related proteins (B). *, mean values significantly larger than that of LRP1^+/− or B41 cells (p < 0.05); **, mean values significantly smaller than that of LRP1^+/− or B41 cells (p < 0.05).

FIGURE 9.

PDGF-mediated ERK phosphorylation and cell proliferation requires LRP1 in mouse fibroblasts. A, differential ERK phosphorylation after PDGF stimulation in LRP1^+/−, LRP1^−/−, and B41 MEFs is shown. All cells were serum-starved overnight then stimulated with PDGF at the indicated concentrations for 15 min at 37 °C. Total cell lysates with phospho-specific ERK IgG (rabbit) are shown. ERK phosphorylation levels were compared with the total ERK protein using specific mouse IgG on the same membrane by using multiplexed infrared detection (Odyssey System). B, shown is the time course of differential ERK phosphorylation during continuous PDGF stimulation. After overnight starvation, cells were induced with 30 ng/ml PDGF-β at 37 °C. At the indicated times, cell lysates of each cell line were prepared and analyzed for phosphor-ERK and total ERK as in panel A. C, LRP1^+/−, LRP1^−/−, and B41 MEFs were tested for PDGF-stimulated cell proliferation. Serum-starved quiescent cells were stimulated with 30 ng/ml PDGF-β at 37 °C. After 24 h cells were counted with hemacytometer. Experiment was performed in triplicate. *, mean of LRP1^+/− significantly larger than that of LRP1^−/− (p < 0.01); **, mean of B41 significantly larger than that of LRP1^−/− (p < 0.001).