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  • The collagen binding membrane proteins discoidin domain rece

    2019-10-08

    The collagen-binding membrane proteins, discoidin domain receptors 1 and 2 (DDR1 and DDR2) belong to the family of receptor tyrosine kinase and are expressed in a variety of mammalian cells.7, 8 These transmembrane glycoproteins (∼125 kDa) have been found to be over-expressed or atypically expressed in several malignancies,9, 10, 11, 12 and regulated in diseases such as atherosclerosis, lymphangioleio-myomatosis, rheumatoid arthritis, and osteoarthritis. DDRs are characterized by three distinct regions: an extracellular domain (ECD), which is responsible for collagen binding, a transmembrane region and an intracellular kinase domain. Binding of collagen(s) to the DDR ECD is known to induce tyrosine phosphorylation of the DDR kinase domain;7, 8 prolonged activation of the DDR kinase domain results in upregulation or activation of matrix metalloproteases (MMPs 1, 2, 9 and 13), which cleave and degrade the collagen fibers in the ECM.7, 14, 16 A second mode of collagen regulation reported earlier by our laboratory shows that the ECD of DDR1 or DDR2 when expressed as a soluble protein can modulate fibrillogenesis of collagen type 1 in-vitro.18, 19 In particular, we found that DDR2 ECD delays collagen fibrillogenesis and the collagen fibers formed in the presence of DDR2 ECD were thinner and lacked the native D-periodic banded structure. However, these earlier observations were based mainly on using purified collagen and a soluble form of DDR2 ECD, whereas thus far the DDR2 ECD has only been reported as an integral component of the membrane-anchored, full-length DDR2 receptor.
    Results
    Discussion We demonstrate here that the DDR2 receptor, even when lacking its kinase domain, still remains a regulating factor in collagen fibrillogenesis by cells. Our investigations reveal that the cell surface aminopeptidase inhibitor of DDR2/-KD results in collagen fibers in the ECM that are deficient in the native banded structure, have smaller fiber diameter, exhibit delayed kinetics for collagen fibrillogenesis and a reduced collagen deposition in the ECM. These observations are consistent with our earlier results, where we showed that the soluble DDR2 ECD when present in collagen solution in-vitro, resulted in a lack of native banded structure, smaller fiber diameters and a delayed kinetics of collagen fibrillogenesis. Our current results thus signify that cell surface-anchored, collagen-binding proteins also have the capacity to regulate collagen fibrillogenesis. We thus elucidate a novel functional role for the expression of DDR2 ECD, which occurs in the full-length DDR2 protein or may be present in other unidentified DDR2 isoforms. Our results may also provide novel insights into the functional roles of isoforms of DDR1. It is known that DDR1 can exist in five distinct isoforms in vivo, DDR1a–e, obtained through alternative splicing. Since our DDR2/-KD construct resembles the naturally occurring kinase-dead DDR1 splice variants, DDR1d and DDR1e, and we showed earlier that the soluble DDR1 ECD regulates collagen fibrillogenesis; it is likely that even the DDR1d and DDR1e isoforms along with the full-length DDR1 have a functional role in collagen regulation. Although the splice variants for DDR2 have not been characterized, there is evidence to support their existence. Several protein species for DDR2 have been detected in cultured human smooth muscle cells at various molecular masses: 130 kDa, 90 kDa, 50 kDa and 45 kDa, along with two transcripts at 9.5 kb and 4.5 kb. Independent studies have identified multiple transcripts for DDR2 in cancerous and normal cell lines.,22, 23, 24 Our results signify the importance of identifying and characterizing the DDR2 isoforms that possess the DDR2 ECD. In addition, our results suggest that other collagen-binding membrane proteins, like integrins and platelet glycoprotein VI, may influence collagen fibrillogenesis, especially if their soluble domains have been demonstrated to regulate collagen fibril formation.