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First published online 18 January 2005
doi: 10.1242/jcs.01402

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Dynamics and interaction of caveolin-1 isoforms with BMP-receptors

Anja Nohe^1,*, Eleonora Keating¹, T. Michael Underhill², Petra Knaus³ and Nils O. Petersen^1,

¹ Department of Chemistry, University of Western Ontario, Chemistry Building, London, N6A 5B7, Canada
² School of Medicine and Dentistry, University of Western Ontario, Health Sciences Addition, Room HSA 110, London, N6A 5C1, Canada
³ Department of Physiological Chemistry, University of Würzburg, 97074 Würzburg, Germany

View larger version (49K): [in a new window]   Fig. 1. BRII is upregulated when A431 cells are serum-starving. Serum-starved or non-serum-starved A431 cells were fixed and fluorescently labeled to test for the expression of (B,C,G) BRII or (D,E,F) BRIa, using a polyclonal antiserum against BRII and a secondary donkey anti goat RRX antibody, respectively. Cells were fixed and high magnification images of flat membrane regions were collected with a confocal microscope. (F,G) Fluorescence-intensities for quantification, calculated from a large number (40) of images from different cells. Zoom-1 image of non-serum-starved A431 cells (A) labeled for BRIa, (B) stained for BRII. (C) Serum starvation of A431 cells leads to upregulation of BRII at the cell surface. (D) Non-serum-starved A431 cells that express BRIa. (E) Starved cells express comparable amounts of BRIa at the cell surface. (F) Expression levels of BRIa are the same for non-starved (N BRIa), starved (S BRIa) and stimulated (S BRIa + B) A431 cells. (G) Expression levels of BRII are very low for non-starved (N BRII) A431 cells, but is increased many-fold after starvation for 72 hours (S BRII) and remains constant upon stimulation with BMP-2 (S BRII + B). (H) A typical autocorrelation function for BRIa.

View larger version (14K): [in a new window]   Fig. 2. Upon BMP-2 stimulation, caveolin-1 ß moves into caveolae enriched in caveolin-1 . A431 cells were either cultured normally in DMEM with 10% FBS (N) or were serum-starved (S). After 3 days, starved cells were stimulated (S+B), or not stimulated (S) with BMP-2, fixed and labeled for (B) caveolin-1 [] or (A) caveolin-1 ß [ß] with monoclonal antibodies against the caveolin-1 isoforms and a secondary fluorescently labeled antibody against the caveolin-1 antibodies. Cell membrane expression was visualized by confocal microscopy. From 40 different cells 40 high-magnification images of the membrane were collected and the average intensity of the labeled caveolin-1 was calculated. For each image, the cluster density (CD) of each of the caveolin-1 isoforms was calculated by ICS (see Materials and Methods) and expressed as a ratio relative to the CD observed for the antibodies recognizing caveolin-1 and ß (C).

View larger version (20K): [in a new window]   Fig. 3. Redistribution of BRII in A431 cells. Colocalization of BMP receptors with caveolin-1 isoforms. A431 cells were either cultured in DMEM with 10% FBS (N) or were serum-starved (S). A431 cells cultured with serum were transfected with BRII (N +BRII) or not. After 3 days starved cells were stimulated with BMP-2 (S+B)or not stimulated (S). Cells were than fixed with paraformaldehyde-saponin (P) or acetone-methanol; BMP-receptors were labeled using polyclonal antisera against BRIa or BRII and a secondary fluorescently labeled antibody. The same cells were labeled for caveolin-1 using an antibody specific for the isoform [] or for both and ß isoforms [ß] and a secondary fluorescently labeled antibody. Cell membrane expression was visualized by confocal microscopy and 40 images were collected at the highest magnification from 40 different cells. From these images the fraction of BMP receptors colocalizing either with caveolin-1 or with and ß were calculated using ICCS. (A) The percent colocalization of BRIa with both isoforms of caveolin-1 [] (black bars) or with both isoforms (all the caveolae) as well as ß alone [ß] (white bars). (B) The percent colocalization of BRII with caveolae with both isoforms [] (black bars) and those with both isoforms and those with mainly ß [ß] (white bars).

View larger version (48K): [in a new window]   Fig. 4. BRII binds to caveolin-1 and caveolin-1 ß. COS-7 cells were co-transfected with plasmids encoding BRII-HA or BRIa-myc and one of the two caveolin isoforms, caveolin-1 or ß. After 48 hours, cells were lysed and proteins were immunoprecipitated as indicated. The immunoprecipitated proteins were subjected to SDS PAGE and transferred onto nitrocellulose. The nitrocellulose membrane was incubated with an antibody recognizing caveolin-1 and ß, the HA or myc-epitopes, and a secondary HRP conjugeated antibody. The top panel shows that the interactions with the caveolin isoforms is specific for BRII and that there are no direct interactions between BRIa and the caveolin-1. The two bottom panels show that BRIa is present in the transfected cells.

View larger version (41K): [in a new window]   Fig. 5. Caveolin-1 interacts with BRIa and BRII in starved A431 cells. A431 cells were serum-starved for 72 hours were then stimulated with BMP-2 or left in medium for 2 hours as indicated. Cells were lysed and BRIa, BRII, the transferrin receptor (TR) and caveolin-1 (cav) were immunoprecipitated with specific antibodies against the proteins. The interaction with caveolin-1 was tested by western-blot. Caveolin-1 interacts with BRII and BRIa in starved A431 cells. After stimulation with BMP-2 these interactions are disrupted.

View larger version (10K): [in a new window]   Fig. 6. Caveolin-1 ß inhibits BMP signaling. Serum-starved A431 cells were transfected with the pSBE and caveolin-1 isoforms as indicated. Cells were stimulated or not with BMP-2 for 24 hours, lysed and reporter gene activity was measured.

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