Abstract
Platelet-derived growth factor C (PDGF-C) is one of four members in the PDGF family of growth factors, which are known mitogens and survival factors for cells of mesenchymal origin. PDGF-C has a unique two-domain structure consisting of an N-terminal CUB and a conserved C-terminal growth factor domain that are separated by a hinge region. PDGF-C is secreted as a latent dimeric factor (PDGF-CC), which undergoes extracellular removal of the CUB domains to become a PDGF receptor alpha agonist. Recently, the multidomain serine protease tissue plasminogen activator (tPA), a thrombolytic agent used for treatment of acute ischemic stroke, was shown to cleave and activate PDGF-CC. In this study we determine the molecular mechanism of tPA-mediated activation of PDGF-CC. Using various PDGF-CC and tPA mutants, we were able to demonstrate that both the CUB and the growth factor domains of PDGF-C, as well as the kringle-2 domain of tPA, are required for the interaction and cleavage to occur. We also show that Arg231 in PDGF-C is essential for tPA-mediated proteolysis and that the released "free" CUB domain of PDGF-C can act as a competitive inhibitor of the cleavage reaction. Furthermore, we studied how the PDGF-C/tPA axis is regulated in primary fibroblasts and found that PDGF-C expression is down-regulated by hypoxia but induced by transforming growth factor (TGF)-beta1 treatment. Elucidating the regulation and the mechanism of tPA-mediated activation of PDGF-CC will advance our knowledge of the physiological function of PDGF-CC and tPA and may provide new therapeutic opportunities for thrombolytic and cardiovascular therapies.
Highlights
TPA-mediated Proteolysis Depends on Both Structural Domains of PDGF-CC—We mapped the structural requirements for recognition of latent PDGF-CC as a substrate for tissue plasminogen activator (tPA) using mutated forms of PDGF-CC in a co-transfection assay
These findings indicate that both the CUB and the growth factor domains are necessary for efficient proteolytic cleavage of latent PDGF-CC by tPA
Until recently little was known about the protease responsible for the activation of PDGF-CC, but we have shown that the fibrinolytic serine protease tPA cleaves and activates PDGF-CC [11]
Summary
Protein-Protein Interaction Studies—To determine which domain(s) of tPA and PDGF-CC are involved in the protein-protein interaction between the two proteins, His6-tagged recombinant PDGF-CC protein species, expressed using the baculovirus expression system as described previously [1], were bound to nickel-nitrilotriacetic acid (Ni-NTA)-agarose (Qiagen) and incubated for 90 min at room temperature with conditioned serum-free media from COS-1 cells transfected with the tPA truncation mutants. As cleavage of PCCUBPD would release the growth factor domain of PDGF-DD, a PDGFR- agonist, the conditioned media were applied to PDGFR- expressing PAE cells, but none induced PDGFR- stimulation.
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