Collagen type I selectively activates ectodomain shedding of the discoidin domain receptor 1: Involvement of Src tyrosine kinase

Abstract

The discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that is highly expressed in breast carcinoma cells. Upon binding to collagen, DDR1 undergoes autophosphorylation followed by limited proteolysis to generate a tyrosine phosphorylated C-terminal fragment (CTF). Although it was postulated that this fragment is formed as a result of shedding of the N-terminal ectodomain, collagen-dependent release of the DDR1 extracellular domain has not been demonstrated. We now report that, in conjunction with CTF formation, collagen type I stimulates concentration-dependent, saturable shedding of the DDR1 ectodomain from two carcinoma cell lines, and from transfected cells. In contrast, collagen did not promote cleavage of other transmembrane proteins including the amyloid precursor protein (APP), ErbB2, and E-cadherin. Collagen-dependent tyrosine phosphorylation and proteolysis of DDR1 in carcinoma cells were reduced by a pharmacologic Src inhibitor. Moreover, expression of a dominant negative Src mutant protein in human embryonic kidney cells inhibited collagen-dependent phosphorylation and shedding of co-transfected DDR1. The hydroxamate-based metalloproteinase inhibitor TAPI-1 (tumor necrosis factor-α protease inhibitor-1), and tissue inhibitor of metalloproteinase (TIMP)-3, also blocked collagen-evoked DDR1 shedding, but did not reduce levels of the phosphorylated CTF. Neither shedding nor CTF formation were affected by the γ-secretase inhibitor, L-685,458. The results demonstrate that collagen-evoked ectodomain cleavage of DDR1 is mediated in part by Src-dependent activation or recruitment of a matrix-or disintegrin metalloproteinase, and that CTF formation can occur independently of ectodomain shedding. Delayed shedding of the DDR1 ectodomain may represent a mechanism that limits DDR1-dependent cell adhesion and migration on collagen matrices. Keywords zinc-dependent metalloproteinase; amyloid precursor protein; tyrosine phosphorylation; TIMP (tissue inhibitor of metalloproteinase)

Shedding of Discoidin Domain Receptor 1 by Membrane-type Matrix Metalloproteinases

“…However, the relative levels of these forms did not match the extent of inhibition of DDR1 shedding observed in the media. This is an agreement with a previous study in which treatment with either the metalloproteinase inhibitor TAPI or TIMP-3 in T47D and DDR1-transfected 293 cells, respectively, had no effect on the levels of full-length or CTF of DDR1, despite a significant inhibition of collagen I-induced DDR1 shedding by these inhibitors (35). In our cell system, we ascribed this paradoxical finding with the TIMPs and MIK-G2 to differences in antibody affinity (sc-532 versus AF2396), technique sensitivity (immunoblot analyses only able to detect drastic shifts in receptor pool), and/or issues of receptor turnover (compensatory mechanisms), all of which may highlight the complex relationship between receptor shedding and generation of degradation fragments (35).…”

“…For instance, the partial inhibition caused by TIMP-1 in HCC1086 cells (Fig. 8A) may be explained by an effect on ADAM10 activity, which is expressed in HCC1806 cells and has been previously suggested to be a potential DDR1 sheddase (35). Thus, we cannot rule out members of the ADAM family of metalloproteinases as additional and important DDR1 sheddases.…”

“…In some surface receptors, the released N-terminal fragment elicits biological activity of its own in the pericellular space, whereas the remnant CTF undergoes intramembranous cleavage followed by translocation to the nucleus where it regulates transcriptional activity (40). Previous studies have shown that DDR1 is shed from the cell surface in both constitutive (41) and collagen-induced manners (35,42), leading to the formation of two C-terminal fragments of 58 and 62 kDa (42). Based on sensitivity to protease inhibitors, it was postulated that DDR1 cleavage is mediated by the action of a metalloproteinase(s) (35).…”

“…Previous studies have shown that DDR1 is shed from the cell surface in both constitutive (41) and collagen-induced manners (35,42), leading to the formation of two C-terminal fragments of 58 and 62 kDa (42). Based on sensitivity to protease inhibitors, it was postulated that DDR1 cleavage is mediated by the action of a metalloproteinase(s) (35). However, the nature of the metalloproteinase(s) was not identified.…”

“…HCC1806 cells also express the mRNA of several secreted and membraneanchored collagenases, including MT1-MMP, MT2-MMP, MMP-1, and MMP-13. In addition, we also examined the expression of ADAM (a disintegrin and metalloproteinase) 10 and 17 mRNAs, two well studied members of the ADAM subfamily of metalloproteinases with established sheddase activity (34), which were also implicated in collagen I-induced shedding of DDR1 in human embryonic kidney 293 cells transfected to express recombinant DDR1 (35). We found that HCC1806 cells express the mRNA of these ADAMs (supplemental Fig.…”