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First published online 25 July 2006
doi: 10.1242/jcs.03074

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Complex biosynthesis of the muscle-enriched iron regulator RGMc

Department of Biochemistry and Molecular Biology, Mail code L224, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97239-3098, USA

View larger version (51K): [in a new window]   Fig. 1. Induction of RGMc mRNA and protein expression during myogenic differentiation. (A) Time course of accumulation of RGMc mRNA and mRNAs for myogenin, myosin heavy chain (MHC) and S17 over 96 hours of differentiation of C2 myoblasts, as measured by RT-PCR. Relative increases in mRNA levels compared with T0 are listed below each time point. (B) Time course of accumulation of RGMc, myogenin and -tubulin over 96 hours of differentiation of C2 myoblasts, as measured by immunoblotting using whole-cell protein extracts. The arrows indicate different immunoreactive RGMc polypeptides. (C) Detection of RGMc in whole-cell protein extracts from C3H10T1/2 cells infected with Ad-ß-gal or Ad-MyoD, and incubated in differentiation medium (DM) for 48 hours. Arrows indicate the immunoreactive RGMc proteins. (D) One major RGMc mRNA species is synthesized in differentiating mouse muscle cells. The upper panel shows a schematic of the mouse RGMc gene. Exons are depicted as boxes, with predicted coding regions in black and introns are indicated by dotted lines. The relative positions of primers and predicted product sizes are displayed below the gene. The lower panel shows results of RT-PCR experiments, using exon-specific primers and RNA from differentiating muscle cells (C2AS12 and C2 myoblasts, 72 hours in DM, Ad-MyoD infected C3H10T1/2 cells, 48 hours). Positions of molecular mass markers are indicated to the left of blots.

View larger version (49K): [in a new window]   Fig. 2. Muscle-cell-associated RGMc is a GPI-anchored membrane protein. (A) Immunocytochemistry for cell-surface associated RGMc (red) in differentiated C2 myotubes (left panel). After pre-absorption of polyclonal anti-RGMc anti-serum with bacterially expressed RGMc bound to CNBr-Sephadex, no binding is seen (right panel). Nuclei in both panels are stained with Hoechst 33258 dye (blue). Magnification, 200x. (B) Detection of RGMc by immunoblotting (arrows) using membranes isolated from differentiated (T72) but not undifferentiated C2 myoblasts (T0), after separation by SDS-PAGE under reducing (upper left panel) and non-reducing conditions (right panel). Equivalent amounts of membrane proteins were used, based on equal detection of cadherin (lower left panel). (C) Detection of cell-surface RGMc by immunoblotting (arrows) after labeling membrane proteins of differentiating C2 myoblasts (72 hours in DM) with a cell-impermeable biotin crosslinking reagent (EZ-link), followed by pull-down of protein extracts with streptavidin-agarose (+, cells exposed to EZ link; -, mock-treated cells). Immunostaining for cadherin is shown below. (D) Detection of muscle RGMc (arrows) released from the surface of differentiated C2 cells (T72) after incubation with (+) or without (-) PI-PLC.

View larger version (31K): [in a new window]   Fig. 3. RGMc is secreted by differentiating muscle cells. (A) Time course of accumulation of RGMc (arrows) in culture media conditioned by differentiating C2 myoblasts (CM). (B) Detection of RGMc by immunoblotting (arrows) in CM from C2AS12 myoblasts after incubation for 72 hours in differentiation medium (DM) with IGF-I but not PDGF (left panel) and by C3H10T1/2 cells after infection with Ad-MyoD but not Ad-ß-gal, and incubation in DM for 48 hours (right panel). (C) Incubation of CM containing RGMc secreted from C2 myoblasts (T72) with PNGase F (+) or after mock treatment (-). White arrowheads indicate digestion products.

View larger version (50K): [in a new window]   Fig. 4. Processing and localization of HA-tagged RGMc. (A) Detection of ectopic cell-surface RGMc by immunoblotting (arrows) in undifferentiated C2 myoblasts infected with either Ad-HA-RGMc (wt) or Ad-HA-RGMcGPI () after labeling membrane proteins with the EZ-link reagent, followed by pull-down of protein extracts with streptavidin-agarose. The left panel shows immunoreactive RGMc in whole-cell protein extracts (WCE); results of pull-down experiments are displayed in the middle and right panels. Antibodies are as indicated. Immunostaining for cadherin is shown below. (B) Detection of RGMc released from the cell membrane following incubation of undifferentiated C2 cells infected with Ad-HA-RGMc with PI-PLC (+). Antibodies are as indicated. WCE is included for reference. For A and B, the gray arrow delineates the C-terminal fragment of RGMc. (C) Digestion of membrane-associated HA-RGMc with PI-PLC followed by PNGase F (+) or mock treatment (-). (D) Digestion of secreted HA-RGMc with PNGase F. Antibodies are as indicated. For C and D, white arrowheads indicate digestion products.

View larger version (106K): [in a new window]   Fig. 5. Overexpression of RGMc or RGMcGPI does not alter muscle differentiation. (A) Immunohistochemistry for ectopic RGMc (anti-HA, green) in C2 myoblasts infected with Ad-tTA and either Ad-HA-RGMc or Ad-RGMcGPI, and incubated in differentiation medium for 48 hours (upper panels). The middle panels show an overlay of immunostaining with anti-HA and anti-myosin heavy chain (MHC; red) from the same microscopic field. Arrows indicate co-staining within multinucleated myotubes. The lower panels show results of immunocytochemistry for RGMc (anti-HA) and myosin heavy chain in C2 myoblasts infected with Ad-tTA and either Ad-HA-RGMc or Ad-RGMcGPI, as described above, and incubated in differentiation medium plus doxycycline for 48 hours (+ dox). Note that the extent of myotube formation and the levels of MHC are comparable whether or not wild-type RGMc or RGMcGPI are overexpressed. Magnification, 200x.

View larger version (47K): [in a new window]   Fig. 6. Biosynthesis and processing of RGMc and RGMcGPI in Cos-7 cells. (A) Time course of accumulation of RGMc in whole-cell protein extracts (WCE) and conditioned culture medium (CM) after infection with Ad-HA-RGMc. Full-length and internally cleaved RGMc proteins are indicated by black or gray arrows, respectively. Immunoblots for tubulin and albumin protein staining (lower panels) indicate equivalent sample loading. (B) RGMc rapidly accumulates at the cell membrane following its biosynthesis. RGMc was analyzed by immunoblotting at the indicated times following infection with Ad-HA-RGMc after labeling of cell membranes with the EZ-link reagent and streptavidin-agarose pull-down. Immunostaining for tubulin and cadherin (lower panels) is included for loading and cell-surface specificity controls, respectively. (C) Time course of accumulation of RGMc in WCE and CM after infection with Ad-HA-RGMcGPI. Full-length and internally cleaved RGMc proteins are indicated by black or gray arrows, respectively. Immunoblots for tubulin and albumin protein staining (lower panels) are included to demonstrate equivalent sample loading. (D) Immunostaining with anti-HA antibody of CM samples from C.

View larger version (47K): [in a new window]   Fig. 7. Differential stability of cell-surface-associated and secreted RGMc proteins in Cos-7 cells. (A) Time course of the decrease in RGMc protein expression in whole-cell protein extracts (WCE) and conditioned culture medium (CM) from Cos-7 cells infected 12 hours earlier with Ad-HA-RGMc and treated with cycloheximide and doxycycline for the times indicated. Immunoblotting for cadherin demonstrates effective inhibition of new cellular protein synthesis and immunoblotting for tubulin and albumin staining show equivalent sample loading (lower panels). Graphical analysis of average band intensity from at least two independent experiments is displayed to the right. (B) Time course of decline in cell-surface RGMc after inhibition of protein synthesis with cycloheximide. Membrane-associated RGMc was labeled by treating cells infected 12 hours earlier with Ad-HA-RGMc with the EZ-link reagent for 30 minutes, followed by addition of cycloheximide and doxycycline for up to 24 hours. Biotin-labeled RGMc or cadherin was detected by immunoblotting following streptavidin-agarose pull-down from WCE. An immunoblot for tubulin (lower panels) is included as a loading control. Average cell-surface expression for two independent experiments of full-length RGMc (fl), RGMc N- and C- terminal fragments (N/C), and cadherin proteins is shown in the graph to the right. (C) Time course of accumulation of biotin-labeled RGMc in conditioned culture medium following treatment of cells with the EZ-link reagent and incubation with cycloheximide and doxycycline for the times indicated. Total and biotin-labeled RGMc in CM was determined by immunoblotting as outlined in B. Average levels for two independent experiments of total and biotin-labeled RGMc are plotted to the right. Black and gray arrows in blots indicate full-length and internally cleaved RGMc proteins, respectively.

View larger version (25K): [in a new window]   Fig. 8. Altered processing of the juvenile hemochromatosis-linked RGMc G313V mutation. (A) Detection of HA-RGMc (WT) and HA-RGMc G313V in whole-cell protein extracts (WCE) and conditioned culture medium (CM) in transiently transfected in Cos-7 cells. Full-length and internally cleaved RGMc proteins are indicated by black or gray arrows, respectively. (B) Detection of RGMc WT and G313V proteins after labeling of cell membranes with the EZ-link reagent and streptavidin-agarose pull down. Immunostaining for tubulin and cadherin (lower panels) is included for loading and cell-surface specificity controls, respectively. (C) Immunostaining with anti-HA antibody of samples from B. In B and C, the full-length, internally cleaved and anomalously processed RGMc proteins are indicated by black, gray or open circles respectively.

View larger version (18K): [in a new window]   Fig. 9. Biosynthesis and processing of muscle RGMc. (A) Diagram of RGMc proteins found on the cell membrane of differentiating skeletal muscle cells and released into the culture medium. Green box, RGD motif; yellow box, partial von Willebrandt factor type D domain; gray stars, asparagine-linked sugars; jagged line, GPI-anchor; S-S, disulfide bonds. (B) Model for biosynthesis and processing of RGMc. The arrows designate different processing paths: (1) intracellular proteolytic processing of RGMc (indicated by red scissors) targeted to the cell membrane by a GPI moiety; (2) full-length, glycosylated RGMc is targeted to the cell membrane by a GPI sequence; release of full-length, glycosylated RGMc from the membrane by a phospholipase (3) or protease (4) (black scissors).

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