Initiation of BMP2 signaling in domains on the plasma membrane.

Bone Morphogenetic Protein 2 (BMP2) is a potent growth factor crucial for cell fate determination. It directs the differentiation of mesenchymal stem cells into osteoblasts, chondrocytes, adipocytes and myocytes. Initiation of BMP2 signaling pathways occurs at the cell surface through type I and type II serine/threonine kinases housed in specific membrane domains such as caveolae enriched in the caveolin-1 beta isoform (CAV1β, caveolae) and clathrin coated pits (CCPs). In order for BMP2 to initiate Smad signaling it must bind to its receptors on the plasma membrane resulting in the phosphorylation of the BMP type Ia receptor (BMPRIa) followed by activation of Smad signaling. The current model suggests that the canonical BMP signaling pathway, Smad, occurs in CCPs. However, several recent studies suggested Smad signaling may occur outside of CCPs. Here we determined; 1) The location of BMP2 binding to receptors localized in caveolae, CCPs or outside of these domains using AFM and confocal microscopy. 2) The location of phosphorylation of BMPRIa on the plasma membrane using membrane fractionation, and 3) The effect of down regulation of caveolae on Smad signaling. Our data indicates that BMP2 binds with highest force to BMP receptors localized in caveolae. BMPRIa is phosphorylated in caveolae and the disruption of caveolae inhibited Smad signaling in the presence of BMP2. This suggests caveolae are necessary for the initiation of Smad signaling. We propose an extension of the current model of BMP2 signaling, in which the initiation of Smad signaling is mediated by BMP receptors in caveolae.