Mol
Mol. 12). However, as Blasticidin S opposed to most RTKs, the kinetics of DDR phosphorylation is usually slow and sustained (4, 5), indicative of a unique activation process that remains to be elucidated. Structurally, the DDRs share a similar basic domain name organization composed of an extracellular N-terminal discoidin domain name (DS) followed by a predicted DS-like domain name (DS-like) (13), a short extracellular juxtamembrane (EJXM) linker, a single-span transmembrane domain name, and an intracellular juxtamembrane domain name connected to a KD (1, 3, 14, 15). For a detailed description of DDR structure and domain name business, see Refs. 1C3. In the case of DDR1, alternative splicing generates five isoforms, which share a similar ectodomain organization. However, these isoforms differ in their intracellular juxtamembrane and KD. For instance, DDR1b and DDR1c contain an additional 37 residues in the intracellular juxtamembrane region (residues 505C541), whereas DDR1c possesses six additional residues in the KD. Thus, these DDR1 variants are fully functional RTKs that may activate different signaling pathways in response to collagen and elicit different cell functions (1C3). In contrast to Blasticidin S DDR1, DDR2 is usually expressed as a single protein species. A unique aspect of DDRs is the nature of their ligand, which includes several collagen types (1). The larger supramolecular structure of collagens does not appear to be critical for DDR activation, as triple-helical peptides bearing the DDR-binding motif are fully capable of inducing receptor phosphorylation. However, collagens are known to undergo multiple structural modifications that alter their mechanical properties, strength, and stability, which are caused in part by the action of membrane-anchored and secreted collagen-degrading proteases, specifically the members of the matrix metalloproteinase family of zinc-dependent endopeptidases (16). In particular, a triad of membrane-type MMPs (MT-MMPs), MT1-(MMP-14), MT2-(MMP-15), and MT3-(MMP-16), are known to be crucial mediators of collagenolysis at the pericellular space (17C19). Because MT-MMPs and DDRs share a common substrate/ligand, we hypothesized that MT-MMPs can regulate collagen-induced activation of DDRs. In this study, we focused on DDR1 and examined the conversation of this receptor with membrane-anchored and secreted collagenases. Although we expected effects on DDR1 activation mediated by collagen degradation, we found that MT1-, MT2-, and MT3-MMP, but not the secreted collagenases, MMP-1 and MMP-13, inhibited DDR1 activation by promoting receptor cleavage at the EJXM region. Our results shed light on a novel conversation between surface proteases and RTKs that integrate collagen-induced signaling and pericellular proteolysis. EXPERIMENTAL PROCEDURES Cell Lines Immortalized monkey kidney epithelial COS1 (CRL-1650) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in Dulbecco’s altered Eagle’s medium FLJ20285 supplemented with 10% fetal bovine serum (FBS), 2 mm l-glutamine, and Blasticidin S antibiotics at 37 C in an atmosphere of 95% air and 5% CO2. Human breast malignancy T47D cells (HTB-133) were obtained from ATCC and cultured in RPMI 1640 medium supplemented with 10% FBS, insulin, and antibiotics. These cells were transfected to stably express human recombinant MT1-MMP, as described previously (20). Human breast malignancy HCC1806 (CRL-2335) cells were obtained from ATCC and cultured in RPMI 1640 medium supplemented with 10% FBS Blasticidin S and antibiotics. Antibodies and Reagents A rabbit polyclonal antibody against the C-terminal region of DDR1 (sc-532) and a mouse monoclonal antibody (mAb) to GAPDH (sc-47724) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Goat polyclonal antibodies against the N-terminal region of DDR1 (AF2396) were from R&D Systems (Minneapolis, MN). mAbs against the catalytic domain name of MT1-MMP (Lem2/15) were a kind gift from Dr. Alicia G Arroyo (Centro Nacional de Investigaciones Cardiovasulares, Madrid, Spain). Anti-phosphotyrosine mAb (clone 4G10?), referred to as anti-Tyr(P), and mAbs against human MT2-MMP (MAB3320), MMP-1 (MAB3307), and MMP-13 (MAB3321) were purchased from EMD Millipore (Billerica, MA). A polyclonal antibody against MT3-MMP (polyclonal antibody 318, directed against residues 318C335 of human MT3-MMP) was produced in our laboratory. Rabbit monoclonal antibodies to human MT4-MMP (EP1270Y) were purchased from Epitomics/Abcam (Burlingame, CA). A mouse mAb against Myc was a nice gift from Dr. Guri Tzivion (University of Mississippi Medical Center, Jackson, MS). Anti-FLAG M2 mAb (F1804) and anti–actin mAb (A5441) were both purchased from Sigma. Anti-TIMP-1 mAb (IM32) was purchased from Calbiochem, and anti-TIMP-2 mAb CA-101 was described previously (21). MIK-G2 (4-[4-(methanesulfonamido)phenoxy]phenylsulfonyl methylthiirane) was synthesized in the Mobashery laboratory, as described previously (22). Rat tail collagen type I (catalog no. 3440-100-01) and mouse collagen IV (catalog no. 3410-010-01) were purchased from Trevigen (Gaithersburg, MD). Collagenase-resistant (is usually 4-hydroxyproline) harboring the DDR recognition motif, GVMFtriple-helical peptide, the DDR1-transfected COS1 cells.