Figure 1.
Confocal plasma membrane localization of α2 ARs and REEPs.
HEK293A cells were transfected individually with either HA-α2A AR, HA-α2C AR, Flag-REEP1, Flag-REEP2, or Flag–REEP6 cDNA. Forty-eight hrs post-transfections, cells were biotinylated in vivo to label plasma membrane proteins. Subsequently, cells were fixed with 4% PFA, but not permeabilized, labeled with various immunofluorescent conjugates, and examined by confocal microscopy. HA- α2 ARs were labeled with 16B12-Alexa 488 (anti-HA) and Flag-REEPs were identified by M2-FITC (anti-Flag) antisera respectively (Left). Plasma membranes were identified by Alexa 594-conjugated avidin (Middle). Merged images are shown (Right). A. α2 AR immunolabeling of non-permeabilized cells demonstrated plasma membrane co-localization of α2A and α2C ARs, as evidenced by co-localization with Alexa 594-conjugated avidin. B. No REEP co-localization was seen with Alexa 594-conjugated avidin, suggesting that REEPs were not localized to the plasma membrane. REEPs were localized intracellularly, within the avidin delineated plasma membrane. REEP immunolabeling of non-permeabilized cells did reveal intracellular staining, despite the absence of permeabilization, due to the ability of antibodies to gain intracellular entry following PFA fixation [27]. Note the absence of immunolabeling of intracellularly localized α2A or α2C ARs, due to the lack of cell permeabilization (as seen in Figure 5). A more complete description of this methodology and findings can be found in Results. Representative of three separate transfections. Scale bars: 25 µm.
Figure 2.
Confocal intracellular immunolocalization of REEPs.
HEK293A cells were transfected with Flag-REEP1, -REEP2, or -REEP6. Forty-eight hrs post-transfection, cells were fixed with 4% PFA, permeabilized, and examined by confocal microscopy. REEPs were stained with M2 antibody (anti-Flag). Golgi and ER compartments were stained for giantin and calreticulin expression respectively. A: REEP1/2/6 (Left) did not co-localize with the Golgi compartment, delineated by the Golgi marker giantin (Middle), as seen in merged images (Right). B: REEP1/2/6 staining (Left) delineated an intracellular reticular pattern that overlapped with the ER marker calreticulin (Middle), as seen in merged images (Right). All REEPs studied identified a reticular pattern more diffuse than that labeled by anti-calreticulin antisera, suggesting a possible alteration in calreticulin distribution upon REEP co-expression (Right). Representative of three separate transfections. Scale bars: 25 µm.
Figure 3.
Confocal ER localization of REEPs with ER Tracker™ dye.
HEK293A cells were transfected with Flag-REEP1, -REEP2, or -REEP6. Forty-eight hrs post-transfection, cells were fixed with 4% PFA, permeabilized, and examined by confocal microscopy. The ER was identified by staining with the ER-specific dye ER Tracker™ Blue/White DPX, which is retained within the ER lumen, thus labeling the ER tubular network (29). REEPs were stained with M2-Alexa 488 antibody (anti-Flag). REEP1/2/6 staining (Left) delineated an intracellular reticular pattern that showed extensive overlap with the ER luminal network (Middle), as seen in merged images (Right). Areas of punctate REEP expression likely represent areas of focal accumulation within the ER and confocal cross-sections of ER tubules. REEP1 demonstrated focal accumulation near the nucleus (arrow), whereas REEP2 was not found in all ER Tracker™-labeled ER regions (arrow), suggesting the existence of possible REEP/ER subdomains. Representative of three separate transfections. Scale bars: 25 µm.
Figure 4.
Confocal REEP isoform co-localization.
HEK293A cells were transfected with HA-REEP1 (carboxyl terminus HA-epitope tag) and either Flag-REEP1, Flag-REEP2, or Flag–REEP6 (carboxyl terminus Flag-epitope tag) cDNA. Forty-eight hrs post-transfections, cells were fixed with 4% PFA, permeabilized, and examined by confocal microscopy. Flag-REEPs (Left) were identified by FITC-M2 (anti-Flag) antisera and HA-REEP1 (Middle) was labeled with Alexa 594 anti-HA antisera. Merged images are shown (Right). Co-expression of HA-REEP1 and Flag-REEP1 showed tremendous overlap, as expected. Flag-REEP2 and HA-REEP1 showed multiple punctate regions of co-expression. However, Flag-REEP2 also exhibited further extensions devoid of HA-REEP1 expression. Lastly, Flag-REEP6 and HA-REEP1 demonstrated a large degree of co-localization. Representative of three separate transfections. Scale bars: 25 µm.
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.
Figure 6.
REEP expression in non-permeabilized HEK293A, NRK, and Rat1 cells.
HEK293A, NRK, and Rat1 cells were transfected with Flag-REEP1, -REEP2, or –REEP6. Forty-eight hrs later, cells were fixed with 4% PFA, but not permeabilized, for immunofluorescent labeling. Flag-REEPs were labeled with M2 (anti-Flag) antisera and identified with Alexa 594-conjugated goat anti-mouse secondary antibody. Note the similar strong peri-nuclear and intracellular reticular staining pattern seen in all three cell lines. Representative of three separate transfections. Scale bars: 10 µm.
Figure 7.
In vivo biotinylation analysis of REEP plasma membrane expression.
To determine if REEPs were expressed at the plasma membrane, HEK293A cells were transfected with Flag-REEP1, Flag-REEP2, or Flag–REEP6 cDNA with or without co-transfected HA-α2A AR or HA-α2C AR cDNAs. Forty-eight hrs post-transfections, cells were treated with the biotinylating reagent EZ-Link Sulfo-NHS-SS-Biotin (Pierce), total cell lysates were isolated, and biotinylated proteins were precipitated by incubation with avidin-agarose. Avidin precipitated proteins and total cell lysates (Input) were then analyzed by SDS-PAGE and immunoblotting techniques. Transferred proteins were probed with monoclonal anti-HA or anti-M2 Ab. Molecular weight markers (kDa) are shown to the left. A. Top: Mature glycosylated α2A and α2C ARs (thick arrow) were predominantly precipitated by avidin, consistent with selective biotinylation of plasma membrane proteins. Aggregated α2 ARs can be seen at the very op of the blot (*). Bottom: No REEPs were precipitated by avidin, demonstrating that they were not present at the plasma membrane, when either expressed with α2 ARs or alone. B. Top: Analysis of total cell lysates for α2A and α2C ARs demonstrated the presence of both mature (thick arrow) and immature forms (thin arrow). Bottom: Immunoblotting of total cell lysates for REEPs is shown, demonstrating strong REEP expression. Representative of three experiments.
Figure 8.
SGMF analysis of REEP membrane localization.
To differentiate plasma and ER membrane localization of REEPs, sucrose gradient membrane fractionation (SGMF) analysis was performed with HEK293A cells transfected with HA-α2C ARs, and Flag-REEP1, -REEP2, or -REEP6. Forty-eight hrs post-transfection, total cell membranes were isolated and separated by layering upon a discontinuous sucrose gradient. Eleven fractions were collected and analyzed by SDS-PAGE and immunoblotting techniques. Top. Plasma and ER membrane fractions were detected by probing with antibodies against Na/K ATPase (A) or calnexin (B) respectively. Fractions containing lighter plasma membranes (#1-4) and heavier ER membranes (#6-11) are demarcated with bars above the fraction number. Molecular weight markers (M) and input loading control lanes (I) were also included. Note progression of α2C ARs (C) from ER to plasma membrane fractions, demonstrating increasing mature glycosylation in plasma membrane and decreasing immature glycosylation in ER membrane fractions (see Figure 10). Bottom. Similar SGMF analysis of REEP1 (D), REEP2 (E), and REEP6 (F) demonstrating that all three REEPs tested were only found in heavier ER, but not plasma, membrane fractions. Mr standards (kDa) are indicated. Representative of three separate transfections.
Figure 9.
Identification of α2 AR and REEP expression in permeabilized and non-permeabilized cells.
HEK293A cells were transfected with HA-α2A AR, HA-α2C AR, Flag-REEP1, Flag-REEP2, or Flag–REEP6 cDNA. Forty-eight hrs post-transfections, cells were fixed with 4% PFA, under permeabilized and non-permeabilized conditions, and examined by wide field immunofluorescent microscopy. HA-α2 ARs were labeled with 16B12-conjugated FITC (anti-HA) antisera and Flag-REEPs were identified by M2-conjugated Alexa 594 (anti-Flag) antisera. Permeabilized: α2 AR staining of permeabilized cells demonstrated plasma membrane and intracellular fluorescence. Note predominant plasma membrane staining, compared to intracellular staining, consistent with efficient plasma membrane trafficking of α2A ARs. α2C AR staining revealed predominant intracellular staining with a perinuclear shadow, due their predominant localization within the ER when expressed in HEK293A cells (25). REEP staining of permeabilized cells showed strong intracellular localization, due to their ER localization. Non-permeabilized: α2A ARs demonstrated extensive staining of the plasma membrane, however, α2C ARs showed staining only of the plasma membrane, demonstrating that the extracellular HA epitope was not accessible by anti-HA antibody under non-permeabilized conditions. Intracellular REEP staining was similar when performed under permeabilized and non-permeabilized conditions, demonstrating that the carboxyl terminal Flag epitope was accessible by anti-Flag antibody under either condition, due to its cytoplasmic localization. REEP staining of non-permeabilized cells revealed intracellular staining, due to the ability of antibodies to gain intracellular entry following PFA fixation [27]. Representative of three separate transfections. Scale bars: 10 µm.
Figure 10.
Representative FACS histograms and REEP gating strategy.
HEK293A cells were co-transfected with HA-α2A or -α2C ARs and control vector, Flag-REEP1, -REEP2, or –REEP6 cDNAs. Forty-eight hrs post-transfection, relative expression levels of each receptor were determined in non-permeabilized (Surface) and permeabilized (Total) cells by using a FACS assay. α2 ARs and REEPs were labeled with FITC-conjugated anti-HA and Cy3-conjugated anti-Flag (M2) antibodies respectively. A. Representative α2A AR FACS fluorescence distributions under non-permeabilized (Top) and permeabilized (Bottom) conditions (UT = untransfected). B. Representative α2C AR FACS fluorescence distributions under non-permeabilized (Top) and permeabilized (Bottom) conditions (UT = untransfected). Note the shift to higher median fluorescence upon permeabilization, which is greater for α2C vs. α2A ARs due to the larger pool of intracellular α2C ARs [25]. C. Representative gating strategy for FACS analysis of co-expressed α2 ARs and REEPs. Background staining of non-transfected HEK293A cells with FITC-conjugated anti-HA and Cy3-conjugated anti-Flag (M2) antibodies was determined (Top) and used to set the FACS gating thresholds for background fluorescence (Q4). Representative α2A AR and REEP1 FACS data set demonstrating co-expression of both proteins is shown (Bottom). All cells contained in quadrants Q1-3 were analyzed for calculation of REEP effects on co-expressed α2 AR surface and intracellular expression (see Table 4A). Data summarized from between five and eight different transfections for each combination of α2 AR and REEP with a minimum of 1000 cells analyzed for each transfection.
Figure 11.
REEP co-expression enhances the presence of a lower molecular weight 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 immunoblot analysis. Molecular weight markers (kDa) are shown to the left. Top: Co-expression of either REEP1, REEP2, or REEP6 with α2C AR correlated with an increased detection of a lower molecular weight form of α2C AR (arrow), not seen following co-expression with α2A ARs. Bottom: Immunoblot analysis of REEPs demonstrated similar levels of REEP1, REEP2, and REEP6 expression when co-expressed with either α2A or α2C ARs. Representative of three experiments.
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.
Figure 13.
Immunoprecipitation of α2 ARs by REEPs.
HEK293A cells were co-transfected with HA-α2A or -α2C ARs and control vector, Flag-REEP1, -REEP2, or –REEP6. 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 rabbit anti-HA Ab (A/C) or anti-M2 (B/D). Molecular weight markers (kDa) are shown to the left. Lanes representing total cell lysates (input), REEP co-IP, and α2 AR co-IP are labeled at the bottom of the blots. A. Note absence of α2A AR co-IP with any REEP tested (middle). B. Similar amounts of REEP1, REEP2, and REEP6 were present in REEP co-IP assays. C. REEP1, REEP2, and REEP6 could co-IP the REEP-enhanced, minimally glycosylated form of α2C AR (Figures 9 and 10). D. As seen with α2A AR/REEP co-IP assays, similar amounts of REEP1, REEP2, and REEP6 were present in REEP co-IP assays. Neither α2A nor α2C ARs could co-IP any REEP tested (B and D, right). IgG light chain artifacts are indicated by an arrow (far right).
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.
Figure 15.
Amino acid comparison of REEP Families.
The REEP family can be subdivided into two subfamilies, REEP1-4, and REEP5-6. Yeast Yop1 is most similar to the latter subfamily and has been included in the alignment. Residues completely conserved in each subfamily are highlighted in yellow, whereas partially similar residues are highlighted in blue (consensus residue derived from a block of similar residues at a given position) or green (consensus residue derived from the occurrence of greater than 50% of a single residue at a given position). Hydrophobic segments are boxed in black. The conserved 14-3-3 binding site (RSXpS) found in REEP1-4 is boxed in red. Conserved positively charged residues postulated to be involved with microtubulin binding in REEP1-4 are demarcated (*), whereas conserved negatively charged residues in REEP5-6/Yop1 are also shown (#) [34]. Alignment performed using Vector NTI v.11 (Invitrogen). GenBank protein accession numbers utilized were:
mouse REEP1 (Yip2a): NP_848723
mouse REEP2 (Yip2d): NP_659114
mouse REEP3 (Yip2b): NP_848721
mouse REEP4 (Yip2c): NP_850919
mouse REEP5 (Yip2e): NP_031900
mouse REEP6 (Yip2f): NP_647453
Yop1: NP_0153