Figure 7.

Mutations within the proximal NPxY motif of the LRP1 tail result in different receptor distributions. (A) FRT cells stably transfected with either mLRP4Y₂₉A or mLRP4N₂₆A were grown to confluence on coverslips and treated for indirect immunofluorescence with anti-HA under nonpermeabilized and permeabilized conditions, as indicated. Y₂₉A distributed both at the apical cell surface (right panel) and intracellular vesicles (left panel), whereas N₂₆A show only an intracellular localization, not being detectable at the cell surface. MDCK cells gave similar results (not shown). Scale bars, 10 μm. (B) Domain-specific cell surface biotinylation of MDCK and FRT cells expressing mLRP4N₂₆A. In MDCK cells the protein was not detected at the cell surface. Controls confirmed the receptor expression (immunoblot of total lysate) and proper basolateral expression of E-cadherin. In FRT cells, however, a low amount of minireceptor was detected at the basolateral cell surface. Na⁺K⁺ ATPase was used as an endogenous basolateral marker. (C) MDCK cells expressing either mLRP4wt or the N₂₆A mutant were consecutively incubated, either intact or after permeabilization with 0.1% saponin with an anti-HA and anti-mouse RPE-conjugated antibody, and analyzed by flow cytometry. The ratio of expression levels observed in intact versus permeabilized cells show 30% of the wild-type receptor versus no more than 4% of the N₂₆A mutant at the cell surface.