REEPs Are Membrane Shaping Adapter Proteins That Modulate Specific G Protein-Coupled Receptor Trafficking by Affecting ER Cargo Capacity
Figure 5
Confocal co-localization of α2 ARs and REEPs in permeabilized cells.
HEK293A cells were co-transfected with HA-α2A or –α2C AR cDNAs and either empty vector (pcDNA3.1), Flag-REEP1, -REEP2, or –REEP6 cDNAs. Cells were fixed with 4% PFA, permeabilized, and examined by confocal microscopy forty-eight hrs post-transfection. α2A and α2C ARs were stained with anti-HA mAb (16B12) and Alexa 594 conjugated-anti mouse secondary antisera; REEPs were stained with rabbit anti-Flag polyclonal antisera and Alexa 488 conjugated anti-rabbit secondary antisera. Confocal images were focused to include ER membrane planes and allow imaging of REEP and α2 AR expression. A. α2A ARs demonstrated predominant plasma membrane expression as described previously [25] (Left). Immunolabeling of REEPs (Middle) identified an intracellular reticular/punctate pattern that did not overlap with plasma membrane localized α2A ARs (Right). B. Immunolabeling for α2C ARs (Left) demonstrated a large intracellular pool of receptor, as described previously [25]. REEPs (Middle) were localized to the intracellular space (ER) and small amount of overlap with α2C ARs was detected (Right). Punctate areas of overlap between α2C ARs and REEPs can be seen, which were not as evident with α2A ARs. Absence of either α2 AR (vector control) did not alter REEP localization. Representative of three separate transfections. Scale bars: 25 µm.