Abstract
The oncogenic roles of PDGF-D and its proteolytic activator, matriptase, have been strongly implicated in human prostate cancer. Latent full-length PDGF-D (FL-D) consists of a CUB domain, a growth factor domain (GFD), and the hinge region in between. Matriptase processes the FL-D dimer into a GFD dimer (GFD-D) in a stepwise manner, involving generation of a hemidimer (HD), an intermediate product containing one FL-D subunit and one GFD subunit. Although the HD is a pro-growth factor that can be processed into the GFD-D by matriptase, the HD can also act as a dominant-negative ligand that prevents PDGF-B-mediated β-PDGF receptor activation in fibroblasts. The active GFD-D can be further cleaved into a smaller and yet inactive form if matriptase-mediated proteolysis persists. Through mutagenesis and functional analyses, we found that the R(340)R(341)GR(343)A (P4-P1/P1') motif within the GFD is the matriptase cleavage site through which matriptase can deactivate PDGF-D. Comparative sequence analysis based on the published crystal structure of PDGF-B predicted that the matriptase cleavage site R(340)R(341)GR(343)A is within loop III of the GFD, a critical structural element for its binding with the β-PDGF receptor. Interestingly, we also found that matriptase processing regulates the deposition of PDGF-D dimer species into the extracellular matrix (ECM) with increased binding from the FL-D dimer, to the HD, and to the GFD-D. Furthermore, we provide evidence that R(340)R(341)GR(343)A within the GFD is critical for PDGF-D deposition and binding to the ECM. In this study, we report a structural element crucial for the biological function and ECM deposition of PDGF-D and provide molecular insight into the dynamic functional interplay between the serine protease matriptase and PDGF-D.
Highlights
Matriptase, the activator of PDGF-D, can abolish its function
HD Has a Dual Function as a Pro-growth Factor and a Dominant-negative Ligand for the -PDGFR—We previously determined that PDGF-D is a substrate of matriptase by incubating Recombinant PDGF-D (rPDGF-D) in conditioned medium (CM) with the purified serine protease domain of matriptase, followed by immunoblot analysis using antiPDGF-D antibody [21]
Consistent with our previous report, the HD (ϳ58 kDa), which consists of a full-length PDGF-D (FL-D) monomer and a growth factor domain (GFD)-M, was detected as an intermediate product under nonreducing conditions before the FL-D dimer was fully processed into GFD dimer (GFD-D)
Summary
Results: The R340R341GR343A motif within the growth factor domain is the matriptase cleavage site through which matriptase destroys PDGF-D activity and binding to the extracellular matrix (ECM). We showed that serine proteases, such as urokinase plasminogen activator and matriptase, can remove the CUB domain from the latent full-length PDGF-D (FL-D) dimer in a stepwise manner, producing a hemidimer (HD) containing one FL-D monomer and one GFD monomer (GFD-M) and subsequently a biologically active GFD dimer (GFD-D) [10, 11]. We previously reported that YR247GR249S (P4 –P1/P1Ј) within the PDGF-D hinge region is the cleavage site for urokinase plasminogen activator or matriptase to remove the inhibitory CUB domain and release the active GFD-D [11].
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