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First published online 25 May 2004
doi: 10.1242/jcs.01156

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A NHERF binding site links the ßPDGFR to the cytoskeleton and regulates cell spreading and migration

¹ Molecular Neurogenetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Building 149, 13th Street, Charlestown, MA 02129, USA
² Schepens Eye Research Institute, Harvard Medical School, 20 Staniford Street, Boston, MA 02114, USA

View larger version (47K): [in a new window]   Fig. 1. NHERF interacts with ßPDGFR in vitro and in vivo in a PDGF-independent manner. (A) Schematic representation of NHERF binding domains. NHERF contains two PSD-95/Dlg/Zo-1 homology (PDZ) domains, protein-protein interaction domains known to associate with specific C-terminal motifs on target proteins. Merlin and ERM (MERM) proteins interact with the 30 amino acids of the C-terminal end of NHERF. (B) Affinity precipitation assays. GST-NHERF fusion proteins encompassing domains PDZ1 (aa 11-97), PDZ1+2 (aa 11-236) and the full-length protein (aa 1-358), affinity precipitate ßPDGFR in glioma 238 cells, whereas PDZ2 alone (aa 149-236) or the C-terminus containing PDZ2 (IC149; aa 149-358), does not. Merlin, but not moesin, is detected in GST-IC149 and full-length NHERF affinity precipitates. (C) The mutant L1106A ßPDGFR does not bind NHERF. Pooled populations of G418-resistant F cells stably expressing the retroviral pLXSN vector (vector), mutant L1106A, wild type (WT) or the kinase-defective mutant K634R, were lysed and assayed for their ability to affinity precipitate with GST-NHERF. (D) Endogenous NHERF immunoprecipitates together with WT but not mutant L1106A ßPDGFR in F cell cultures that were maintained in complete growth medium.

View larger version (64K): [in a new window]   Fig. 2. NHERF binding to ßPDGFR is not essential for PDGF-mediated actin cytoskeletal reorganization. PDGF-BB stimulation (5 ng/ml for 10 minutes) of F cells expressing WT or the mutant L1106A results in the aggregation of F-actin at the cell periphery (arrows), dorsal ruffles (arrowheads), filopodia formation and diminution of actin stress fibers. Scale bars, 10 µm.

View larger version (88K): [in a new window]   Fig. 3. F cells expressing the mutant L1106A exhibit delayed spreading on FN. (A) Serum-deprived F cells expressing vector only, WT, or the mutants L1106A or K634R were plated on FN-coated tissue culture plates in serum-free media and examined for their ability to spread 20, 40 and 90 minutes after plating. (B) F cell spreading-efficiency was quantified by calculating the percentage of spread cells at each time point. Shown is the mean±s.d. of triplicates from at least three independent experiments.

View larger version (110K): [in a new window]   Fig. 4. F cells expressing the mutant L1106A migrate slower in wound healing assays. L1106A expression inhibits F cell migration compared with cells that express vector, WT or the mutant K634R F in wound healing assays. Upon wounding, F cell monolayers were incubated in DMEM supplemented with 0.2% FBS and analyzed for their ability to repopulate the wounded area. Phase-contrast images were taken at 10 and 22 hours after wounding. Assays were conducted at least three times and yielded similar results.

View larger version (89K): [in a new window]   Fig 5. Altered F-actin rearrangement in F cells. (A) F cells expressing the mutant L1106A exhibit altered F-actin rearrangement and focal adhesion formation. F cells, expressing WT ßPDGFR or the L1106A mutant in serum-free medium, were plated on FN-coated coverslips for 90 minutes and fixed. Cells were then stained with anti-paxillin (secondary antibody: Alexa Fluor 488-conjugated goat anti-mouse IgG) to examine focal adhesion formation and with Alexa Fluor 594-conjugated phalloidin to examine the actin cytoskeleton. F cells expressing the mutant L1106A exhibit a nonpolar spreading-morphology, a dense ring of cortical F-actin and an increased number and density of focal adhesions. Scale bars, 10 µm. (B) F cells expressing WT ßPDGFR and the C-terminus of NHERF display delayed spreading on FN. Serum-deprived F cells expressing WT ßPDGFR together with a HA-tagged C-terminal fragment of NHERF (aa 270-358), which encompasses the MERM binding domain and EGFP, or F cells expressing WT ßPDGFR and EGFP alone (vector), were plated on FN-coated coverslips. After 90 minutes, the cells were fixed and stained for F-actin with Alexa Fluor 594-conjugated phalloidin. GFP expression identifies transfected cells. Scale bars, 20 µm.

View larger version (49K): [in a new window]   Fig. 6. FN-mediated FAK tyrosine phosphorylation. (A) Serum-deprived F cells expressing WT or mutant L1106A ßPDGFR were plated on FN-coated cell culture plates for the indicated times. Cell lysates were prepared and analyzed for either FAK Y397 phosphorylation or total FAK tyrosine phosphorylation (4G10). The same blots were detected with a polyclonal FAK antibody to assess FAK levels. An immunoblot representating three independent experiments shows decreased total tyrosine phosphorylation of FAK in cells expressing the mutant L1106A. (B) Quantitative comparison of FAK Y397 and total tyrosine phosphorylation from F cells expressing WT or the mutant L1106A ßPDGFR. Values were obtained from laser scanning densitometry and indicate the relative mean±s.d. from band intensities normalized to total FAK protein of three independent experiments. The asterisk indicates statistical significance (P<0.05).

View larger version (21K): [in a new window]   Fig 7. Affinity precipitation of FAK with merlin and NHERF. (A) GST merlin N-terminus (aa 1-345), but not merlin C-terminus (aa340-590), affinity precipitates FAK. (B) Merlin full-length isoform 1 (aa 1-595) and isoform 2 (aa 1-590) GST fusion proteins, which differ by 16 C-terminal amino acids, associate with FAK in MGF1092 cells. (C) Full-length NHERF-GST fusion protein associates with merlin and FAK in glioma 238 cell lysates.

View larger version (35K): [in a new window]   Fig 8. Model for ßPDGFR-NHERF linking growth factor receptor and integrin signaling (adapted from Sieg et al., 2000). FAK is shown to form a complex with ßPDGFR-EGFR and suggested to be an important link between growth factor receptor and integrin signaling pathways (Sieg et al., 2000). As a direct interaction between FAK and ßPDFR-EGFR could not be demonstrated, it was suggested that one or more intermediary bridging proteins mediate this association. Our results suggest that NHERF and MERM proteins could be the intermediary bridging proteins.

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