REEPs Are Membrane Shaping Adapter Proteins That Modulate Specific G Protein-Coupled Receptor Trafficking by Affecting ER Cargo Capacity
Figure 12
REEP co-expression enhances the presence of a minimally-glycosylated form of α2C ARs.
HEK293A cells were transfected with either HA-α2A or -α2C ARs and control vector, Flag-REEP1, -REEP2, or –REEP6. Forty-eight hrs post-transfection, crude membranes were isolated and subjected to endoglycosidase digestion (C = No Enzyme, E = Endoglycosidase H (Endo H), P = PNGase F (PNGase)). Due to loss of signal during enzymatic digestion, 125 µg of protein was digested and loaded in each lane. Mature (M) and immature (I) glycosylated forms are indicated. Endo H cleaves only immature glycosylated forms, whereas PNGase cleaves all glycosylated forms. Molecular weight markers (kDa) are shown on the left. Note that α2A ARs exhibit mostly mature (Endo H insensitive), whereas α2C ARs show mostly immature (Endo H sensitive), glycosylation patterns, correlating with their predominant plasma membrane and intracellular localizations respectively. The presence of REEP1, REEP2, or REEP6 with either α2 AR did not alter the relative ratios of mature to immature glycosylation. However, co-expression of either REEP1, REEP2, or REEP6 with α2C ARs correlated with an increased expression of a lower molecular weight form that was minimally glycosylated (arrow), not seen following co-expression with α2A ARs. Apparent loss of the minimally glycosylated form after PNGase treatment was due to instability in PNGase enzymatic buffer conditions (data not shown). Representative of three experiments.