3X FLAG tag Several reports by us and others
Several reports by us and others have highlighted that dimerization or oligomerization of DDR1 ECD and DDR2 ECD enhances their binding to triple-helical collagen.18, 19, 22, 28, 29, 30, 31 The ECD of DDRs consists of a discoidin domain and a stalk region. It has been reported that independent 3X FLAG tag of the cytoplasmic domain of DDR1 did not inhibit receptor dimerization. More recently, Abdulhussein et al. found that the cysteine residues, 303 and 348 present in stalk region of DDR1 ECD are essential for receptor dimerization and deletion of the stalk region prevented receptor dimerization. Further, Abdulhussein et al. demonstrated that glutathione S-transferase (GST) tagged DDR1 ECD and DDR2 ECD when expressed as a soluble protein bound to collagen. The molecular mass of the DDR1 ECD-GST and DDR2 ECD-GST proteins was found to be around 62 kDa, similar to the molecular masses of the DDR1/ECD and DDR2/ECD proteins utilized in this study. We have also recently shown that DDR1 exists as a dimer on the cell surface, independent of the presence of collagen, and undergoes further oligomerization upon ligand stimulation. Since the DDR1/ECD and DDR2/ECD proteins in our stable cell lines preserve the capacity to interact with collagen, they are likely expressed as dimers and may undergo further oligomerization upon interaction with collagen. Our results signify that both membrane-anchored and soluble isoforms of DDR ECD proteins may be important in ECM remodeling. In this regard, several soluble collagen-binding proteins secreted in the ECM—decorin,3, 32, 33, 34 lumincan,3, 34, 35 biglycan,34, 36 fibromodulin,34, 35, 36 periostin, aggrecan, and versican—are known to regulate collagen fibrillogenesis. Decorin and lumican are known to regulate collagen fibril diameter,3, 38 and the absence of biglycan and fibromodulin inhibits the maturation of collagen fibrils. Animal studies have begun to reveal the importance of soluble collagen-binding proteins in the regulation of collagen maturation and fiber diameter. Studies on knockout mice for decorin, lumican, fibromodulin,35, 36 and periostin have demonstrated that these proteins are critical to generate a uniform collagen fiber diameter in tissues. For example, ultrastructural analyses of the cornea, skin, and tendon from lumican knockout mice shows collagen fibers with increased fiber diameters, while the tendon from fibromodulin knockout mice contains higher frequency of smaller fiber diameters. Limited studies exist on the effects of membrane-anchored proteins on fibrillogenesis of type 1 collagen. Integrins α5β1 and α2β1 are understood to modulate collagen fibrillogenesis predominantly with fibronectin polymerization as a prerequisite. Although the integrin α1(I) and α2 (I) domains have been shown to affect collagen fibrillogenesis as soluble proteins in vitro, no changes in collagen fiber density or organization were observed in the integrin α1β1 or α2 subunit-deficient mice. Interestingly, the knockout mice for the orphan receptor Gpr48 and the transmembrane collagen XIII showed disrupted collagen fibrils, although the mechanisms of their interaction with collagen type 1 are not well understood. Our current results along with our previous findings indicate that DDR ECDs serve as a robust model system to compare and contrast how membrane-anchored versus soluble proteins may regulate collagen fiber structure and deposition. We had earlier reported that collagen fibers with intact native banded structure were occasionally observed in the kinase-deficient, membrane-anchored DDR2 ECD (DDR2/-KD) samples; however, in our DDR1/ECD and DDR2/ECD samples, observation of native banded structure of collagen was far more infrequent. D-periodicity of collagen fibers from native cultures was measured at 61±5 nm, which is in agreement with previous studies by us and others. Previously, we found that the membrane-anchored DDR2/-KD inhibited lateral fiber growth, compared to native cultures. While fiber diameter measurements for the first week of culture for DDR1/ECD and DDR2/ECD gave results similar to those of DDR2/-KD samples (25.0–28.9 nm), fibers in DDR2/-KD cultures exhibited lateral growth of around 10 nm over the 3 weeks of culture observed. In contrast, a sustained inhibition of lateral growth of collagen fibers was observed by DDR ECD proteins resulting in average collagen fiber diameters between 20 and 30 nm throughout a 4-week period. Together, our results show that soluble DDR2 ECD inhibits collagen fibrillogenesis in the ECM consistent with membrane-anchored DDR2, albeit with a slightly higher potential. We speculate that this stronger inhibition of collagen fiber structure and lateral diameter is due to the soluble DDR ECD being distributed throughout the ECM and thus having more ability to affect collagen fiber formation even in ECM regions away from the pericellular regions.