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First published online May 24, 2006
doi: 10.1242/10.1242/jcs.02946

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Caveolin-1 is required for signaling and membrane targeting of EphB1 receptor tyrosine kinase

Meri M. Vihanto¹, Cecile Vindis^1,*, Valentin Djonov², Douglas P. Cerretti³ and Uyen Huynh-Do^1,

¹ Department of Nephrology and Hypertension, and Department of Clinical Research, Inselspital, University of Bern, CH-3010 Bern, Switzerland
² Institute of Anatomy, University of Bern, CH-3000 Bern, Switzerland
³ Amgen Corporation, 1201 Amgen Court West, Seattle, WA 98101, USA

View larger version (63K): [in a new window]   Fig. 1. Ligand-stimulated Eph receptors interact with caveolin-1. (A) Co-fractionation of EphB1 receptor with caveolin-1. Sucrose gradient centrifugations were performed as described previously (Song et al., 1996a). Fractions were collected and subjected to immunoblotting (IB) with antibodies directed against HA-tagged EphB1 receptor (Anti HA) and caveolin-1 (Anti Cav-1). EphB1 receptor and caveolin-1 located to the same, buoyant membrane fractions (#1-3). Immunoblotting with antibodies directed against Src (Anti c-Src) was used to show localization of a cytoplasmic protein, as it was located to the high-density fractions (#6-8). (B) Co-immunoprecipitation (IP) of EphB1 receptor with antibody against caveolin-1 in CHO-EphB1 cells. EphrinB2/Fc was added to the cells for indicated times, then cells were lysed, caveolin-1 (lanes 3-7) and control rabbit IgG (lanes 1-2) immunoprecipitates were resolved by SDS-PAGE and transblotted to PVDF membranes. Membranes were reciprocally immunoblotted with antibodies against HA or caveolin-1, respectively. (C) Immunofluorescence analysis confirmed the association of EphB1 receptor with caveolin-1 on the plasma membrane after 30 minutes of ephrinB2 ligand stimulation (C2) compared with non-stimulated cells (C1). CHO-EphB1 cells were fixed and stained with primary antibodies against HA and caveolin-1 followed by Alexa green- and red-conjugated secondary antibodies, respectively. Samples were processed as described under Materials and Methods. Images were acquired with a Nikon Eclipse E600 microscope, connected to Nikon digital camera DXM1200, and processed with the Nikon AC-1 software version 2.11. Magnification, x1000. (D) Co-immunoprecipitation of EphA2 receptor with antibody against caveolin-1 in PC-3 cells. EphrinA1/Fc was added to the cells for indicated times, then cells were lysed, caveolin-1 (lanes 1-5) and control rabbit IgG (lanes 6-7) immunoprecipitates were resolved by SDS-PAGE and transblotted to PVDF membranes. Membranes were reciprocally immunoblotted with antibodies against EphA2 or caveolin-1, respectively. Results are representative of at least three independent experiments.

View larger version (60K): [in a new window]   Fig. 2. EphrinB2 stimulates tyrosine phosphorylation of caveolin-1 at Tyr14. (A) CHO-EphB1 cells were serum starved for at least 24 hours, then stimulated for indicated times with 1-2 µg/ml ephrinB2/Fc. Cell lysates were separated by SDS-PAGE, transferred to PVDF membranes, and blotted with antibodies against phospho-caveolin (p-cav, upper panel) or caveolin-1 (cav-1, lower panel). (B) Immunofluorescence analysis confirmed the increasing tyrosine phosphorylation of caveolin-1 up to 60 minutes after ephrinB2 ligand stimulation (B2) compared with non-stimulated cells (B1). Thereafter, the phosphorylation of caveolin-1 decreased steadily. CHO-EphB1 cells were fixed and stained with primary antibody against phospho-caveolin followed by Alexa red-conjugated secondary antibody. Samples were processed as described under Materials and Methods. Images were acquired with a Nikon Eclipse E600 microscope, connected to Nikon digital camera DXM1200, and processed with the Nikon AC-1 software version 2.11. Bars, 20 µm. (C) CHO cells were serum starved for at least 24 hours, then stimulated for indicated times with 1-2 µg/ml ephrinB2/Fc. Cell lysates were separated by SDS-PAGE, transferred to PVDF membranes, and blotted with antibodies against phospho-caveolin (upper panel) or caveolin-1 (lower panel). Results are representative of at least three independent experiments.

View larger version (59K): [in a new window]   Fig. 3. Cholesterol depletion inhibits activation of the ERK/MAPK pathway by EphB1. (A) CHO-EphB1 cells were incubated with 0 (Fig. A1,2) or 10 mM (Fig. A3,4) ß-cyclodextrin (ß-CD) for 60 minutes, when cells were prepared for electron microscopy as described under Materials and Methods. Transmission electron microscopy (Fig. A, panels 1 and 3) and scanning electron microscopy (Fig. A, panels 2 and 4) reveal caveolae structures on the surface of the untreated CHO-EphB1 cells, indicated by arrowheads (insert in Fig. A1). Bars, 3 µm (panels 1,3), 12 µm (panels 2,4), and 5 µm (inserts in panels 2,4). A single cell is shown in the inserts shown in panels 2 and 4. (B,C) CHO-EphB1 cells were incubated with or without 10 mM ß-CD for 60 minutes, and then stimulated with 1-2 µg/ml ephrinB2 ligand for 30 minutes. (B) Co-immunoprecipitation (IP) of EphB1 receptor with antibody against caveolin-1 in CHO-EphB1 cells. The immunoprecipitates were resolved by SDS-PAGE and transblotted to PVDF membranes. Membranes were reciprocally immunoblotted with antibodies against HA or caveolin-1, respectively. (C) The state of ERK phosphorylation was determined by immunoblotting with a phosphospecific antibody against active ERK (P-Erk). Stripped membranes were then reblotted with antibody against ERK (Erk1/2). Relative phosphorylation levels are given as a ratio of phosphorylated protein expression to unphosphorylated protein expression. Protein expressions were analyzed using Image J software (http://rsb.info.nih.gov/ij/). Results are representative of at least three independent experiments.

View larger version (39K): [in a new window]   Fig. 4. Characterization of EphB1 mutants lacking the caveolin-binding motif. (A) Relative mRNA expression of EphB1 mutant and wild-type (wt) receptors was detected using real-time RT-PCR (numbers correspond to mutant and wt receptors listed at the bottom of the figure; for further details of the mutants, see text). The total RNA isolation was performed using Trizol® reagent. The 18S steady-state mRNA level was used as reference. The results showed that all of the EphB1 receptor constructs were expressed at the mRNA level. (B) ELISA was performed using the ephrinB2/Fc chimera to detect EphB1 expression on the cell membrane. The binding of the chimera was detected using AP-conjugated secondary antibody and color reaction was initiated by the addition of p-nitrophenyl phosphate. There was no detectable cell-surface expression of any of the mutant receptors when compared with CHO cells transfected with wild-type EphB1 receptor or with CHO-EphB1 cells. Results are representative of at least three independent experiments.

View larger version (102K): [in a new window]   Fig. 5. EphB1 mutants lacking the caveolin-binding motif fail to localize to the cell membrane. The following were fixed and stained with anti-HA and anti-caveolin-1 primary antibodies followed by Alexa green- and red-conjugated secondary antibodies, respectively: CHO-EphB1 (O,P) and untransfected CHO cells (A,B); and CHO cells transiently transfected with wild-type EphB1 (C,D), mutant W808A (E,F), mutant Y810A (G,H), mutant W815F (I,J), triple mutant clone 1 (K,L), and triple mutant clone 14 (M,N). Samples were processed as described under Materials and Methods. The images are shown in two dimensions (A,C,E,G,I,K,M,O) and in three dimensions (B,D,F,H,J,L,N,P). Slides were viewed by laser-scanning confocal microscope with a x63 objective. (Q) The percentage of EphB1 receptors localized on the membrane for each of the mutant EphB1 receptor constructs, as well as for non-transfected CHO cells (CHO), CHO cells transfected with wild-type receptor (wt-EphB1) and CHO-EphB1 cells, were calculated semi-quantitatively from representative cells and presented as % of the total number of the EphB1 receptors (n=3). (R-S) Examples of three-dimensional images (CHO-EphB1 cells and Y810A mutant, respectively) that were used for receptor counting and localization. The x, y and z axis are indicated in the images. Several cross-sections of each individual cell were used to localize the receptors in the three-dimensional images in Imaris 4.1.3. Software. Results are representative of at least three independent experiments.

View larger version (51K): [in a new window]   Fig. 6. The caveolin-1 scaffolding domain is required for interaction of EphB1 with caveolin-1 and for ERK activation by EphB1. (A,B) Expression of caveolin-1 in Cos-7 cells, Cos-7 cells transfected with wild-type caveolin-1 (Cav-wt), and CHO-EphB1 by immunoblotting (A) and by immunofluorescence (B1-B3, respectively). (B) The cells were fixed and stained with anti-caveolin-1 primary antibody followed by Alexa red-conjugated secondary antibody. Samples were processed as described under Materials and Methods. Images were acquired with a Nikon Eclipse E600 microscopy, connected to Nikon digital camera DXM1200, and processed with the Nikon AC-1 software version 2.11. Bars, 20 µm. (C) Cos-7 cells were transiently transfected with empty vector, wild-type (Cav-wt) or mutant caveolin-1 (Cav-mut) constructs, and additionally with wild-type EphB1 receptor: non-transfected Cos-7 cells (lane 1), empty vector (lanes 2-3), Cav-wt construct (lanes 4-5), and Cav-mut construct (lanes 6-7). Cells were co-transfected with wild-type EphB1 receptor (lanes 2-7). After 48 hours, the cells were incubated for 30 minutes with 1-2 µg/ml ephrinB2/Fc. Upper panel, co-immunoprecipitation of EphB1 receptor with anti-caveolin-1 antibody. The immunoprecipitates were resolved by SDS-PAGE and transferred to PVDF membranes. Membranes were probed with anti-HA or anti-caveolin-1, respectively. Lower panel, ephrinB2/Fc-induced ERK phosphorylation following transfection with the mentioned constructs was assessed using a phosphospecific antibody, then stripped blots were reprobed with anti-ERK antibody. (D) Cos-7 cells were transiently transfected with Cav-wt or Cav-mut constructs and additionally with wild-type or mutant EphB1 receptor: non-transfected Cos-7 cells (D1), Cos-7 cells transfected with wild-type EphB1 receptor and Cav-wt (D2), mutant EphB1 receptor and Cav-wt (D3), wild-type EphB1 receptor and Cav-mut (D4), and mutant EphB1 receptor and Cav-mut (D5). The cells were fixed and stained with anti-HA and anti-caveolin-1 primary antibodies followed by Alexa green- and red-conjugated secondary antibodies, respectively. Samples were processed as described under Materials and Methods. Images were acquired with a Nikon Eclipse E600 microscopy, connected to Nikon digital camera DXM1200, and processed with the Nikon AC-1 software version 2.11. Magnification, x1000. Results are representative of at least three independent experiments.