fluvastatin Multiple protein species are known to
Multiple protein species are known to naturally exist for the transmembrane receptors DDR1 and DDR2. Five splice variants have been characterized for DDR1 (“a” through “e”). The d and e isoforms lack the intracellular kinase domain of DDR1. The splicing of DDR1 to various extents has been reported in human ovarian cancer, breast cancer, and fetal brain and has thus far been best characterized in colon cancer cell lines. In normal and diseased arteries of nonhuman primates, three isoforms (a, b, and d) have been detected; these isoforms are differently expressed in advanced atherosclerotic lesions and have been detected in normal human lung tissue and cultured human smooth muscle cells (SMCs). Although splice variants for DDR2 have not yet been characterized, there is ample evidence to support they exist. In one report, Northern blot analysis using a cDNA probe corresponding to the ECD of DDR2 has revealed multiple mRNA species in melanoma carcinoma and virus-transformed normal embryonic lung cell lines. In a separate study, a major 10-kb transcript and a minor 4.5-kb transcript for DDR2 were detected in human and rat heart and other tissues using Northern analysis, along with additional weak bands at 0.8, 3.6, 2.4, and 1.7 kb. In mouse and rat heart tissue, a DDR2 probe hybridized to multiple RNAs of varying lengths (∼4 and 7 kb). At least two transcripts for DDR2 (9.5 and 4.5 kb) and several protein species (130, 90, 50, and 45 kDa) have been found in cultured human SMCs using fluvastatin against DDR2 ECD. Besides alternate splicing of DDRs, shedding of the DDR1 ECD as a soluble protein in the ECM is another naturally occurring phenomenon reported for several mammalian cells.13, 14 While no direct evidence for DDR2 ECD shedding exists, the Western blots with DDR2 antibodies on SMCs transiently transfected with full-length DDR2 show several smaller molecular species (90, 50, and 45 kDa) besides the full-length 130-kDa receptor, suggesting the likelihood of shedding of DDR2 ECD. Our current investigations highlight the relevance of further characterizing DDR2 isoforms and understanding the different functional roles of DDR1 and DDR2 protein variants. 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 deletion 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.