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First published online 15 February 2005
doi: 10.1242/jcs.01674

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CD98 modulates integrin ß1 function in polarized epithelial cells

¹ Division of Nephrology, Departments of Medicine, Vanderbilt University Medical Center, 1161 21st Avenue South, Nashville, TN 37232, USA
² Cell and Developmental Biology, Vanderbilt University Medical Center, 1161 21st Avenue South, Nashville, TN 37232, USA
³ Cancer Biology, Vanderbilt University Medical Center, 1161 21st Avenue South, Nashville, TN 37232, USA
⁴ Department of Research Medicine, Veterans Affairs Hospital, 1310 24th Avenue South, Nashville, TN 37232, USA

View larger version (27K): [in a new window]   Fig. 1. The transmembrane and juxtamembrane domains of CD98 are highly conserved. (A) Amino acid sequences of the cytoplasmic, transmembrane and proximal extracellular domains of the human, mouse, hamster, zebrafish (DR) and Drosophila (DM) CD98 protein or CD98 homologs. Zebrafish and human CD98 had 44 identical amino acids out of 47 (94%) in the transmembrane and juxtamembrane domains but only 32% identity for the rest of the protein (data not shown). (B) The sequence of the Drosophila homolog accession number AY070626 was compared to the human CD98 sequence at 10-amino acid intervals utilizing alignment software MultAlin (Corpet, 1988). The percentage of sequence similarity was plotted on the y axis and the amino acid number on the x axis.

View larger version (24K): [in a new window]   Fig. 2. Generation of stable IMCD cell populations expressing CD98 or CD98/CD69 chimeras. IMCD cells were transfected with the constructs illustrated. Transfected cell populations were sorted by flow cytometry utilizing antibodies to either the human CD98 or the human CD69 extracellular domain. The expression levels of the cell populations (solid lines) are shown relative to IMCD cells transfected with pcDNA3 vector only (dotted lines). C, cytoplasmic domain; T, transmembrane domain; E, extracellular domain.

View larger version (17K): [in a new window]   Fig. 3. The transmembrane domain of CD98 is required to enhance cell adhesion and migration. (A) The IMCD cell populations transfected with the constructs indicated were plated onto 96-well plates coated with collagen I at the concentrations indicated for 1 hour in serum-free medium. Adherent cells were then stained with crystal violet, lysed and the OD measured. Data represent the mean±s.d. of quadruplicate samples/cell population. (B) IMCD cell populations were plated in transwell dishes coated with 1 µg/ml collagen I and migration was evaluated 4 hours after plating. The values indicate the mean±s.d. of three independent experiments. Differences in cell adhesion and cell migration between CD98 or CD98 chimeras and vector control cells (*), or between C69T98E98 and CD98 (#), or CD98 and C98T98E98 (+) were significant with P<0.05.

View larger version (32K): [in a new window]   Fig. 4. The transmembrane domain of CD98 associates with ß1 integrin. (A) Equal amount of cell lysates (500 µg) from cell populations transfected with either control vector (Vector) or the constructs illustrated were immunoprecipitated with an anti-human CD98 (upper panel) or an anti-mouse ß1 integrin (middle panel) antibody. The immunoprecipitates were separated by 7% SDS-PAGE and transferred to nitrocellulose. Membranes were subsequently immunoblotted with an anti-mouse integrin ß1 (upper panel) or an anti-human CD98 (middle panel) antibody. The lower panel represents an immunoblot of total cell lysates for mouse ß1 integrin to confirm equal loading. (B) Cell lysates from cell populations prepared as described in A were immunoprecipitated with an anti-human CD69 (upper panel) and immunoblotted with an anti-mouse integrin ß1. The lower panel is an immunoblot of the cell lysates for mouse integrin ß1 to demonstrate equal loading.

View larger version (19K): [in a new window]   Fig. 5. Generation of stable IMCD cell populations expressing CD98 or CD98 deletion mutants. IMCD cells were transfected with the full-length or deletion CD98 constructs illustrated. The cell populations were sorted by flow cytometry utilizing an antibody to the human CD98 extracellular domain. The expression levels of the cell populations (solid lines) are shown relative to IMCD cells transfected with vector only (dotted line)

View larger version (11K): [in a new window]   Fig. 6. The five N-terminal amino acids of the transmembrane domain of CD98 are required for increased cell adhesion and migration. (A) The different IMCD cell populations transfected with the CD98 constructs indicated were plated on collagen I at the concentrations indicated as described in Fig. 3. The values represent the mean±s.e. of quadruplicate samples/cell population. (B) Migration assays were performed as described in Fig. 3. Bars and errors represent the mean±s.e. of three independent experiments. Differences in cell adhesion and migration between CD98 or CD98 truncations and vector control cells (*) or between CD98-82 and CD98 (#) were significant with P<0.05.

View larger version (80K): [in a new window]   Fig. 7. The five N-terminal amino acids of the transmembrane domain of CD98 are required for CD98 association with ß1 integrin. (A) Equal amount of cell lysates (500 µg) from cell populations transfected with either control vector or the CD98 constructs indicated were immunoprecipitated with an anti-human CD98 (upper panel) or an anti-mouse ß1 integrin (third panel). The immunoprecipitates were separated by SDS-PAGE as indicated in Fig. 4 and membranes were incubated with an anti-mouse integrin ß1 (upper panel) or an anti-human CD98 antibody (third panel). Equal amounts of cell lysate were immunoblotted with an anti-human CD98 (second panel) or an anti-mouse integrin ß1 antibody (fourth panel) to demonstrate equal amounts of CD98 and ß1 integrin in the different cell populations.

View larger version (31K): [in a new window]   Fig. 8. CD98 association with integrin ß1 induces FAK and AKT phosphorylation. The different cell populations transfected with the CD98 constructs indicated were serum starved for 12 hours, trypsinized and left in suspension or replated on 10 µg/ml collagen I for 10 or 30 minutes. Equal amounts of cell lysate were separated by 10% SDS-PAGE and transferred to nitrocellulose. The membranes were immunoblotted with antibodies to phospho-FAK (p-FAK), total FAK, phospho-AKT (p-AKT), total AKT, phospho-ERK (p-ERK) and ERK.

View larger version (35K): [in a new window]   Fig. 9. CD98-induced cell adhesion and migration is mediated by PI 3-kinase. The cell populations transfected with the CD98 constructs indicated were serum starved for 12 hours and then kept untreated or treated with Wortmannin (100 nM) or LY294002 (5 µM) for 4 hours. The cells were trypsinized and their adhesion (A) and migration (B) analyzed as described in Fig. 3. To verify that Wortmannin and LY294002 inhibited PI3-K activity, cells cultured with or without the inhibitors were lysed 10 minutes after plating on collagen I and immunoblotted for phospho-AKT and total AKT (40 µg lane). Differences in adhesion and migration between untreated and Wortmannin- or LY294002-treated cells (*) were significant with P<0.05.

View larger version (51K): [in a new window]   Fig. 10. CD98 association with integrin ß1 induces focal adhesion formation. IMCD populations expressing the different constructs indicated were plated onto collagen I-coated coverslips for 1 hour and cells were subsequently stained with Alexa 594-conjugated phalloidin to visualize the cytoskeleton (upper panel) and anti-phosphopaxillin antibody to identify activated focal adhesions (middle panel). The lower panels show merged images. Bar, 50 µm.

View larger version (42K): [in a new window]   Fig. 11. CD98 association with integrin ß1 alters IMCD cell branching morphogenesis. IMCD populations expressing the different constructs indicated were grown in 3-D collagen I gels for 9 days. Phase-contrast images of typical examples of branching structures are shown. Bar, 50 µm.

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