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Fig. 9. LRP1 deficiency and impairment of endocytosis increase extracellular transglutaminase activity and cell adhesion by upregulation of cell-surface tTG. Surface tTG levels (A), extracellular transglutaminase activity (B) and adhesion to the 42-kDa fragment of fibronectin (C) were determined for normal (MEF) and Lrp1^–/– (PEA-13) mouse embryonic fibroblasts lacking or expressing tTG (left panels) and for wild-type CHO cells, either untreated or treated with 2 µM bafilomycin or 35 µM chloroquine for 16 hours (right panels). (A) LRP1 deficiency and inhibition of endocytosis upregulate the levels of cell-surface tTG. The steady-state surface tTG levels were determined by surface labeling with sulfo-NHS-LC-biotin, isolation of biotinylated proteins, SDS-PAGE and immunoblotting, and expressed as percentages of the total tTG levels. The results are representative of three independent experiments performed for each cell type (means ± s.d.). (B) The absence of LRP1 and ablation of endocytosis increase the enzymatic activity of tTG on the cell surface. Cell-mediated incorporation of ³H-putrescine into N,N-dimethylcaseine was measured as described previously (Belkin et al., 2001). (C) The lack of LRP1 and treatment with endocytic inhibitors enhance adhesion of cells to the tTG-binding 42-kDa fragment of fibronectin (Radek et al., 1993). Adhesion to the immobilized 42-kDa fragment was measured as described previously (Akimov et al., 2000). The data in (B,C) are means of two independent experiments performed in triplicate.

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