REEPs Are Membrane Shaping Adapter Proteins That Modulate Specific G Protein-Coupled Receptor Trafficking by Affecting ER Cargo Capacity
Figure 14
REEP1 HSP mutant Arg113X does not interact with α2C ARs.
HEK293A cells were co-transfected with HA-α2C AR and either Flag-REEP1 (WT) or Flag-REEP1 (HSP), an HSP mutant form of REEP1 with a premature stop codon at Arg113, leading to a loss of the carboxyl terminus. Eighteen hrs post-transfection, total cell lysates were isolated and analyzed by co-immunoprecipitation (co-IP) with M2 antibody (anti-Flag) and immunoblotting. Transferred proteins were probed with anti-HA Ab or anti-M2 antibodies. Molecular weight markers (kDa) are shown to the left. Inputted total cell lysates were probed for either α2C ARs (A) or REEP (B). Note smaller size of HSP mutant REEP1, consistent with the loss of the carboxyl terminus, and lower expression levels seen following transfection. Following REEP co-IP, WT REEP1, but not HSP mutant REEP1, was able to immunoprecipitate the minimally glycosylated form of α2C AR (C). REEP co-IP immunoblot is shown (D). Representative of three experiments.