BMP9 is produced by hepatocytes and circulates mainly in an active mature form complexed to its prodomain

Bone Morphogenetic Protein 9 (BMP9) has been recently found to be the physiological ligand for the activin receptor-like kinase 1 (ALK1), and to be a major circulating vascular quiescence factor. Moreover, a soluble chimeric ALK1 protein (ALK1-Fc) has recently been developed and showed powerful anti-tumor growth and anti-angiogenic effects. However, not much is known concerning BMP9. This prompted us to investigate the human endogenous sources of this cytokine and to further characterize its circulating form(s) and its function. Analysis of BMP9 expression reveals that BMP9 is produced by hepatocytes and intrahepatic biliary epithelial cells. Gel filtration analysis combined with ELISA and biological assays demonstrate that BMP9 circulates in plasma (1) as an unprocessed inactive form that can be further activated by furin a serine endoprotease, and (2) as a mature and fully active form (composed of the mature form associated with its prodomain). Analysis of BMP9 circulating levels during mouse development demonstrates that BMP9 peaks during the first 3 weeks after birth and then decreases to 2 ng/mL in adulthood. We also show that circulating BMP9 physiologically induces a constitutive Smad1/5/8 phosphorylation in endothelial cells. Taken together, our results argue for the role of BMP9 as a hepatocyte-derived factor, circulating in inactive (40%) and active (60%) forms, the latter constantly activating endothelial cells to maintain them in a resting state.     

Superactive mutants of thromboxane prostanoid receptor: functional and computational analysis of an active form alternative to constitutively active mutants

In class A GPCRs the E/DRY motif is critical for receptor activation and function. According to experimental and computational data, R3.50 forms a double salt bridge with the adjacent E/D3.49 and E/D6.30 in helix 6, constraining the receptor in an inactive state. The disruption of this network of interactions facilitates conformational transitions that generate a signal or constitutive activity. Here we demonstrate that non-conservative substitution of either E129^(3.49) or E240^(6.30) of thromboxane prostanoid receptor (TP) resulted in mutants characterized by agonist-induced more efficient signaling properties, regardless of the G protein coupling. Results of computational modeling suggested a more effective interaction between G_q and the agonist-bound forms of the TP mutants, compared to the wild type. Yet, none of the mutants examined revealed any increase in basal activity, precluding their classification as constitutively active mutants. Here, we propose that these alternative active conformations might be identified as superactive mutants or SAM.