Figure 5.

The presence of the SNX17-binding domain or LRP1 in the megalin tail increases the expression of the chimeric receptor at the cell surface. (A) Schematic representation of megalin minireceptors. The sequences shown in B, corresponding to the native or mutated SNX17-binding domain of LRP1, were inserted proximal to the transmembrane domain of megalin, generating the constructs mMeg-2 and mMeg-3, respectively. mMeg-1 is a megalin minireceptor that harbors the fourth ligand-binding domain and the transmembrane and cytoplasmic domains of megalin (23) (B) Sequences of the SNX17-binding domain of LRP1 and the point of insertion within the megalin cytoplasmic domain. (C) Control or SNX17 KD HEK293 cells were transiently transfected with the minireceptors mMeg-1, mMeg-2, and mMeg-3. The cells (either intact or permeabilized with 0.05% saponin) were then incubated with anti-HA and Alexa-488-conjugated anti-mouse IgG and analyzed through flow cytometry. The results are plotted as the ratio of the minimegalin expression levels observed in intact versus permeabilized cells. The assays were performed in triplicate, and the averages ± SD are plotted in the graph (*p < 0.01 versus mMeg-1).