Reciprocal requirement for balanced expression of GPIbα and filamin for efficient trafficking to the plasma membrane. (A) Overexpression of filamin traps GPIbα in the ER and prevents its redistribution to the plasma membrane. Stable HEK293T cell lines were established in which filamin A was either knocked-down ∼ 90% (KDF), expressed at normal levels (N), or overexpressed 2 to 3-fold (OXF), as shown by filamin immunoblot analysis in the top panel. Each of these 3 cell lines were then transfected with a cDNA encoding GPIbα, and the ability of GPIbα to traffic from the ER to the cell surface evaluated by immunoblot analysis using an antibody that recognizes the extracellular domain of GPIbα. As shown in the bottom panel, cells expressing excess filamin (OXF) accumulated noticeably more GPIbα in the ER, as reported by the persistence of a lower, immature, Endo H-sensitive form of the subunit. Note that similar transfection efficiency was observed when GFP expression vector was transfected into these 3 cell lines. (B) Forced accumulation of GPIbα within the ER traps filamin and prevents its redistribution to the membrane skeleton of the cell. HEK293T cells that stably express FlnA-DsRed were transiently transfected with GPIbα-EGFP alone or together with GPIbβ and GPIX expression plasmids. Colocalization of GPIbα (green) with filamin A (red) was analyzed by confocal laser scanning microscopy. Top 3 panels: GPIbα, when expressed with the other subunits of the GPIb complex, becomes efficiently expressed on the cell surface, and chaperones filamin to the inner face of the plasma membrane. Bottom 3 panels: when expressed without GPIbβ and GPIX, GPIbα becomes largely trapped in the ER, trapping filamin as well, suggesting that they need to associate in nearly equimolar amounts for efficient trafficking of both components to the cell surface.