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First published online 26 June 2003
doi: 10.1242/jcs.00519

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Effect of the distribution and clustering of the type I A BMP receptor (ALK3) with the type II BMP receptor on the activation of signalling pathways

Anja Nohe1, Eleonora Keating1, T. Michael Underhill2, Petra Knaus3 and Nils O. Petersen1,*

1 Department of Chemistry, University of Western Ontario, London, N6A1B7, Canada
2 School of Dentistry, Faculty of Medicine and Dentistry, University of Western Ontario, London, N6A1B7, Canada
3 Department of Physiological Chemistry, University of Würzburg, 97074 Würzburg, Germany



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  		Fig. 1. Co-expression of BRII and BRIa leads to rearrangement of BRIa on the cell surface. Stimulation of cells expressing BRII and BRIa with BMP-2 causes an aggregation of the receptors. BRIa-ca, which is constitutively active, needs the co-expression of BRII for redistribution. COS-7 cells were transiently transfected with myc-tagged BRII and HA-tagged versions of BRIa or BRIa-ca. After 48 hours, cells stimulated with BMP-2 or nonstimulated cells were fixed and the receptors labelled with fluorescent antibodies against the epitope tags. Cell membrane expression of the receptors was visualised by confocal microscopy. Forty images were collected at the highest magnification (Zoom10). From these images the CD value of the BMP receptors was calculated using ICS. (A) COS7 cells were mock transfected, fixed and incubated with the fluorescent HA-antibody. The mock-transfected cells showed no significant fluorescence. (B) COS7 cells were mock transfected, fixed and incubated with the biotin conjugated myc antibody followed by the secondary anti-biotin RRX antibody. The mock-transfected cells show no significant fluorescence. (C) COS7 cells were transfected with a plasmid encoding HA-BRIa, fixed and fluorescently labelled using the FITC conjugated HA-antibody. (D) COS7 cells were transfected with a plasmid encoding myc-BRII. Cells were fixed and fluorescently labelled using the biotin-conjugated myc-antibody followed by a RRX conjugated anti-biotin antibody. (E) Calculated average CD values for BRIa or BRIa-ca. Transfected COS7 cells were fluorescently labelled with a fluorescent antibody against the HA-epitope. Confocal images were taken and the CD values for BRIa and BRIa-ca were calculated by applying ICS. (F) Calculated average CD values for myc-BRIa. After transfection cells were fixed and labelled using a biotin-conjugated antibody against the myc-epitope followed by a fluorescent secondary antibody. Confocal images were taken and the CD values for myc-BRIa were calculated by applying ICS. (G) Calculated average CD values for myc-BRII. Transfected COS7 cells were fluorescently labelled with a biotin-conjugated antibody against the myc-epitope followed by a fluorescent anti-biotin antibody. Confocal images were taken and the CD values for BRII were calculated by applying ICS.

 


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  		Fig. 2. Co-expression of BRII-myc and HA-BRIa-ca leads to an increase in stimulation of the Col2-luc reporter gene activity compared with the single transfection of BRIa-ca or BRII. Co-transfection of BRIa-ca with BRII-KR does not lead to an increase in luciferase activity indicating that the kinase activity of BRII is required for signalling. Limb mesenchymal cells (isolated from day 11.5 mouse embryos) were transfected with a Col2-luc reporter, and expression constructs for BRII, BRIa-ca and pRLSV40 for normalisation. Cells were plated at high density in the wells of a 24-well tissue culture dish, and 48 hours following transfection, cells were lysed and luciferase activity measured. The Col2-luc reporter is derived from the promoter and enhancer regions of the type 2 collagen gene and provides a readout on the status of chondroblast differentiation. BMPs as well as BRIa-ca are known to promote chondrogenesis in limb mesenchymal cells.

 


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  		Fig. 3. Co-expression of BRII-myc and HA-BRIa-ca leads to an increase in stimulation of the pSBE-Luc reporter gene activity compared with the single transfection of BRIa-ca. BRIa-ca and BRII-KR do not increase the luciferase activity of the pSBE-Luc. Also co-expression of BRIa-ca and BRII-KR does not result in an increase in luciferase activity, indicating that the kinase activity of BRII is required. Limb mesenchymal cells (isolated from day 11.5 mouse embryos) were transfected with a pSBE-Luc reporter, expression vector containing either of the two receptors and pRLSV40 (normalisation). Cells were plated at high density in the wells of a 24-well tissue culture dish, and 48 hours following transfection cells were lysed and luciferase activity measured.

 


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  		Fig. 4. Expression of BMP receptors in A431 cells. (A) A431 cells upregulate BRII mRNA expression upon serum starvation. RNA was prepared from starved and nonstarved A431 cells and reverse transcribed. Using specific primer combinations for amplification of BRIa (primer BRIa-9 and BRIa-15) or BRII (primer BRII-15 and BRII-25) by PCR the presence of the receptors in the cells was tested. A431 cells express the mRNA for BRIa in both starved and nonstarved conditions, whereas BRII mRNA is upregulated in starved cells. (B,C) Serum starvation of A431 cells leads to upregulation of BRII at the cell surface. Normal (B) or serum starved A431 cells (C) were fixed and fluorescently labelled for the expression of BRII using a polyclonal antiserum against BRII and a secondary donkey anti-goat RRX antibody. Cells were fixed and images collected using a confocal microscope. (D) Normal A431 cells were fixed and fluorescently labelled for BRIa using a polyclonal antibody against BRIa followed by RRX-conjugated secondary antibody. (E) Serum starved A431 cells were fixed and fluorescently labelled for BRIa using a polyclonal antibody against BRIa followed by RRX-conjugated secondary antibody.

 


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  		Fig. 5. BRII and BRIa-ca are both necessary for the activation of the Smad pathway. A431 cells were transfected with various combinations of BRIa, BRII, BRIa-ca and BRII-KR, and the pSBE-Luc. For normalisation, the renilla luciferase construct pRLSV40 was co-transfected and the emission measured. Only co-transfection of BRIa-ca with BRII or co-transfection of BRIa and BRII with addition of BMP-2 leads to activation of the reporter. The data indicate that BRII is necessary for the activation of the Smad pathway.

 


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  		Fig. 6. CD and intensity (I) of BMP receptors in A431 cells. A431 cells were cultured either in DMEM with 10% FBS or under serum starved conditions. Cells were grown in DMEM without FBS for 72 hours, then stimulated with BMP-2 or not stimulated. The cells were then fixed and the receptors labelled using polyclonal antisera against (A) BRIa and (B) BRII, and a secondary fluorescently labelled antibody. Cell membrane expression of receptors was visualised by confocal microscopy. Forty images were collected at the highest magnification. From these images the CD value of the receptors was calculated using ICS. The CD value of the background fluorescence caused by nonspecific labelling of the antibody was subtracted.

 

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© The Company of Biologists Ltd 2003