Direct Binding and Activation of Receptor Tyrosine Kinases by Collagen

Abstract

Receptor tyrosine kinases play an important role in the control of cell growth, differentiation, metabolism, and cell migration among many other biological responses. To date, more than sixteen families of receptor tyrosine kinase have been identified. The first receptor tyrosine kinases were identified as the cell surface receptors that transduce the intracellular signals of growth factors, such as epidermal growth factor (EGF) or hormones such as insulin (13Schlessinger J Ullrich A Neuron. 1992; 9: 383-391Abstract Full Text PDF PubMed Scopus (1281) Google Scholar). All receptor tyrosine kinases exhibit similar molecular architecture and are activated by a common mechanism. This large receptor family is characterized by an extracellular ligand-binding domain, a single transmembrane region, and a cytoplasmic domain that contains a conserved catalytic protein tyrosine kinase core and sequences responsible for positive and negative receptor regulation (13Schlessinger J Ullrich A Neuron. 1992; 9: 383-391Abstract Full Text PDF PubMed Scopus (1281) Google Scholar). A general mechanism for ligand-induced activation of receptor tyrosine kinases has been established (11Lemmon M.A Schlessinger J Trend Biochem.Sci. 1994; 19: 459-463Google Scholar), in which ligand binding to the extracellular domain induces receptor dimerization. Receptor dimerization in turn leads to activation of the catalytic protein tyrosine kinase domain and to tyrosine autophosphorylation. Receptor autophosphorylation is mediated by an intermolecular process: each protomer phosphorylates the neighboring receptor molecule. Tyrosine autophosphorylation provides an important regulatory role. Phosphorylation of tyrosines within the catalytic domain is essential for maintaining the tyrosine kinase in an active state, while phosphorylation of tyrosine residues that are located in noncatalytic regions leads to generation of docking sites for SH2 (Src homology 2) and PTB (phosphotyrosine binding) domains of signaling molecules (12Pawson A Nature. 1995; 373: 573-579Crossref PubMed Scopus (2202) Google Scholar). A variety of signaling proteins are directly recruited by activated receptors while other signaling molecules are activated by tyrosine phosphorylation (13Schlessinger J Ullrich A Neuron. 1992; 9: 383-391Abstract Full Text PDF PubMed Scopus (1281) Google Scholar, 12Pawson A Nature. 1995; 373: 573-579Crossref PubMed Scopus (2202) Google Scholar). In addition to its regulatory role, receptor oligomerization provides a simple mechanism for generation of signal diversity as growth factor–induced receptor homo- and heterodimerization leads to the recruitment and activation of different complements of intracellular signaling molecules (11Lemmon M.A Schlessinger J Trend Biochem.Sci. 1994; 19: 459-463Google Scholar). The conservation of the amino acid sequences of the catalytic domain of receptor tyrosine kinases enabled development of various strategies for cloning of a variety of novel receptor tyrosine kinases. In most cases the identity of the physiological ligands of these new receptors was not known and hence these receptors were designated “orphan” receptors. In recent years the physiological ligands of several orphan receptors were identified. The classical paradigm is that ligands of growth factor receptors are soluble proteins usually found in blood or other body fluids. Such soluble growth factors activate their specific cell surface receptors by multivalent interactions (Figure 1) and exert their biological effects by endocrine, paracrine, or autocrine mechanisms. Indeed, several ligands of “orphan” receptor tyrosine kinases were found to be soluble proteins that circulate in the blood or other body fluids (9Klein R Jing S Nanduri V O'Rourke E Barbacid M Cell. 1991; 65: 189-197Abstract Full Text PDF PubMed Scopus (1122) Google Scholar, 18Stitt T.N Conn G Gore M Lai C Bruno J Radziejewski C Mattson K Fisher J Gies D.R Jones P.F et al.Cell. 1995; 80: 661-690Abstract Full Text PDF PubMed Scopus (583) Google Scholar). However, more recently a number of membrane-bound ligands have been described. Studies of these new molecules, as well as recent studies of more traditional soluble ligands, have led to the identification of novel variations of ligand receptor interactions responsible for receptor dimerization and activation (Figure 1). Fibroblast growth factors represent a large class of growth factors that cannot activate their surface receptors without the cooperation of accessory molecules. FGF1 or FGF2 bind to FGF-receptors (FGFR) monovalently and are therefore unable to promote receptor dimerization and tyrosine kinase activation (14Schlessinger J Lax I Lemmon M Cell. 1995; 83: 357-360Abstract Full Text PDF PubMed Scopus (444) Google Scholar). Oligomerization of FGF molecules is mediated via multimeric interactions with soluble or membrane-attached heparin sulfate proteoglycans allowing FGF to induce FGF receptor dimerization and tyrosine kinase activation (Figure 1). Indeed, in intact cells heparin sulfate proteoglycans are required for FGF stimulation of FGF receptor dimerization, tyrosine kinase activation, and signaling via FGF-receptors (16Spivak-Kroizman T Lemmon M.A Dikic I Ladbury J.E Pinchasi D Huang J Jaye M Crumly G Schlessinger J Lax I Cell. 1994; 79: 1015-1024Abstract Full Text PDF PubMed Scopus (586) Google Scholar). Another class of ligands were shown to be nondiffusable proteins, that are stably associated with plasma membranes. This family of ligands activate cognate receptors that are expressed on the surface of neighboring cells, thus providing a mechanism for cell–cell communication between different cell types (2Beckmann M.P Cerretti D.P Baum P Venden-Bos T James L Farrah T Kozolsky C Hollingsworth T Shilling H Maraskovsky E et al.EMBO J. 1994; 13: 3757-3762Crossref PubMed Scopus (146) Google Scholar, 3Cheng H.S Flanagan J.G Cell. 1994; 79: 157-168Abstract Full Text PDF PubMed Scopus (324) Google Scholar, 5Davis S Gale N.W Aldrich T.H Maisonpierre P.C Lhotak V Pawson T Goldfarb M Yancopoulos G.D Science. 1994; 266: 816-819Crossref PubMed Scopus (622) Google Scholar). A large family of membrane bound proteins called Ephrins have been identified as the ligands for the EPH receptors: the largest family of receptor tyrosine kinases. The A class of Ephrins are attached to the cell membrane by means of a glycosylphosphatidylinositol (GPI) linkage while the B class of Ephrins are transmembrane proteins composed of an extracellular EPH-binding domain, a transmembrane region, and a cytoplasmic domain. Each class of Ephrins binds a unique subset of receptors that are accordingly designated A or B receptors. This classification represents most of Ephrin/EPH receptor interactions, although some interactions cross the boundaries between the two groups. The binding of Ephrin to EPH receptors on neighboring cells causes a repulsive signal that is essential for correct axonal navigation in the nervous system. It was recently demonstrated that the binding of EPH receptor to a TM (transmembrane) Ephrin leads to tyrosine phosphorylation of the cytoplasmic domain of the Ephrin, suggesting that a bidirectional signal is being transduced in cells expressing the TM-Ephrin (6Holland S.J Gale N.W Mbamau G Yancopoulos G.D Henkemeyer M Pawson T Nature. 1996; 383: 722-725Crossref PubMed Scopus (453) Google Scholar). Yet, another paradigm was revealed through studies of Ret—a receptor tyrosine kinase that is activated by glial-derived neurotrophic factor (GDNF). In this particular case the receptor tyrosine kinase functions as a regulatory subunit of a complex containing a separate membrane-attached ligand-binding subunit. Thus, GDNF binds to a GPI-linked GDNF-receptor (GDNFR) that forms a complex in the plane of the membrane with Ret (7Jing S Wen D Yu Y Holst P Luo Y Fang M Tamir R Antonio L Hu Z Cupples R Louis J Hu S Altrock B Fox G Cell. 1996; 85: 1113-1123Abstract Full Text Full Text PDF PubMed Scopus (1031) Google Scholar). The binding of GDNF induces dimerization of GDNFR/Ret complexes leading to tyrosine kinase activation and stimulation of intracellular signaling pathways (Figure 1). Two reports in the December issue of Molecular Cell (15Shrivastava A Radiziejewski C Campbell E Lubomir K McGlynn M Ryan T.E Davis S Goldfarb M.P Glass D.J Lemke G Yancopoulos G.D Mol. Cell. 1997; 1: 25-34Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar, 19Vogel W Gish G.D Alves F Pawson T Mol. Cell. 1997; 1: 13-23Abstract Full Text Full Text PDF PubMed Scopus (750) Google Scholar) describe an intriguing new paradigm for activation of receptor tyrosine kinases by a new family of ligands (Figure 1 and Figure 2). A search for ligands that activate the “orphan” receptors DDR1 and DDR2 resulted in a surprising observation; the extracellular matrix protein collagen binds to and activates these two receptor tyrosine kinases. The two receptors, designated DDR1 and DDR2, are characterized by a structural domain of 160 amino acids in their extracellular part that exhibits strong sequence similarity to the Dictyostelium discoideum protein discoidin 1 (17Springer W.R Cooper D.N.W Barondes S.H Cell. 1984; 39: 557-564Abstract Full Text PDF PubMed Scopus (130) Google Scholar, 8Johnson J.D Edman J.C Rutter W.J Proc. Natl. Acad. Sci. USA. 1993; 90: 5677-5681Crossref PubMed Scopus (115) Google Scholar), coagulation factors V and VIII, and to a Xenopus laevis recognition protein, A5. Discoidin 1 is a lectin with binding specificity toward galactose and N-acetyl galactosamine and is essential for Dictyostelium discoideum cell aggregation (17Springer W.R Cooper D.N.W Barondes S.H Cell. 1984; 39: 557-564Abstract Full Text PDF PubMed Scopus (130) Google Scholar). DDR1 and DDR2 were independently isolated by many laboratories from human, mouse, and rat tissues (see references in15Shrivastava A Radiziejewski C Campbell E Lubomir K McGlynn M Ryan T.E Davis S Goldfarb M.P Glass D.J Lemke G Yancopoulos G.D Mol. Cell. 1997; 1: 25-34Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar, 19Vogel W Gish G.D Alves F Pawson T Mol. Cell. 1997; 1: 13-23Abstract Full Text Full Text PDF PubMed Scopus (750) Google Scholar). DDR1 is primarily expressed in epithelial cells in the brain, gastrointestinal tract, lung, and kidney, while DDR2 is expressed in brain, heart, and in muscle tissues. Interestingly, DDR1 was found to be overexpressed in mammary, ovarian, and lung carcinomas, suggesting that this receptor may play a role in the tumorigenesis of epithelial carcinomas (1Alves F Vogel W Mossie K Millauer B Hofler H Ullrich A Oncogene. 1995; 10: 609-618PubMed Google Scholar). The two papers by 19Vogel W Gish G.D Alves F Pawson T Mol. Cell. 1997; 1: 13-23Abstract Full Text Full Text PDF PubMed Scopus (750) Google Scholar and 15Shrivastava A Radiziejewski C Campbell E Lubomir K McGlynn M Ryan T.E Davis S Goldfarb M.P Glass D.J Lemke G Yancopoulos G.D Mol. Cell. 1997; 1: 25-34Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar present convincing evidence that DDR1 and DDR2 receptors bind specifically to different types of collagen. DDR1 and DDR2 are differentially activated by various collagens. While DDR1 is activated by collagen type I, II, III, V, and XI, DDR2 is activated mainly by collagen type I and III. The activation of DDR1 and DDR2 by collagen is direct and tyrosine phosphorylation of the two receptors can be stimulated by soluble fibrillar collagen and by collagen immobilized on tissue culture dishes. Since heat denaturation abrogated the ability of collagen to stimulate DDR1 activation and gelatin was unable to stimulate receptor tyrosine phosphorylation, it was concluded that the triple-helical structure of collagen is essential for DDR receptor activation. It was also shown that collagen-induced activation of DDR2 receptor was resistant to pepsin treatment and sensitive to collagenase treatment, which cleaves before the X or Y residue of the Gly-X-Y repeat. The primary structure of collagen is composed of numerous stretches of Gly-X-Y repeats that coil around each other to form a left-handed triple helix. The residue in X position is frequently a proline while Y is frequently a 4-hydroxyproline residue. The inability of collagenase-treated collagen to activate the DDR receptor indicates that an intact Gly-X-Y repeat is essential for receptor activation. In addition, periodate treatment of collagen, which partially removes glycoconjugates, compromised the ability of collagen to stimulate tyrosine phosphorylation of DDR2 receptor, suggesting that carbohydrates bound to collagen may play a role in DDR receptor recognition and activation. It was demonstrated that binding of collagen to DDR1 leads to tyrosine phosphorylation and recruitment of the docking protein Shc. Moreover, activation of DDR2 by collagen enhances the expression of matrix metalloproteinase 1 (MMP1). Both reports conclude that DDR1 and DDR2 are novel signaling receptors for collagen. The kinetics of DDR receptor activation by collagens is very different from the kinetics of activation of most receptor tyrosine kinases by their specific ligands. Growth factors such as EGF or PDGF stimulate receptor activation within seconds both in vitro and in living cells. By contrast, tyrosine phosphorylation of DDR receptors was detected only after prolonged treatment (30–60 min) and phosphorylation was sustained for as long as 16 hr. The temporal dependence of DDR receptor activation is unique; it may indicate that receptor activation is mediated by a rate-limiting step that is determined by the weak interaction and slow on-rate for binding of collagen to these two receptors. It is now well-established that collagen and other components of the extracellular matrix play an important role in the control of cell shape, cell migration, cell proliferation, and differentiation. The primary cellular receptors for collagens are α2β1 or α1β1 integrins (10Kuhn K Eble J Trends Cell Biol. 1994; 4: 256-261Abstract Full Text PDF PubMed Scopus (109) Google Scholar). Analysis of signaling pathways that are activated by engagement of integrins by extracellular matrix proteins has demonstrated a remarkable similarity to the signaling pathways activated by growth factor receptors (4Clark E.A Brugge J.S Science. 1995; 268: 233-239Crossref PubMed Scopus (2782) Google Scholar). This includes the canonical Ras/MAP kinase pathway, a signaling pathway mediated by PI-3 kinase and by Akt/PKB, as well as activation of phospholipase Cγ and phosphoinositide metabolism, among other intracellular events. There is good evidence for synergy between signaling pathways that are activated by growth factors and signaling pathways downstream of integrins. In addition, it is well known that normal growth control requires both anchorage dependence and tightly regulated signaling via growth factor receptors. The realization that collagen, one of the most abundant proteins in vertebrates, can activate a family of receptor tyrosine kinases, provides further evidence that signaling via integrins and receptor tyrosine kinases is regulated in a coordinated fashion. It is proposed that collagen binds to DDR receptors in a fundamentally different manner than growth factor binding to their receptors. It is possible that the low-affinity interaction between collagen and DDR receptors is secondary to collagen's primary interaction with integrins. According to this scenario, DDR receptors would function as signaling components of a multi-integrin complex that transmits the intracellular signal initiated at the cell surface by engagement of integrins by collagen and perhaps also other extracellular matrix proteins (Figure 2). It is noteworthy that an association between αvβ3 integrin and IRS1, a major substrate of insulin receptor, was detected in lysates of insulin-stimulated cells (20Vuori K Ruoslahti E Science. 1994; 266: 1576-1578Crossref PubMed Scopus (338) Google Scholar). These two reports provide convincing evidence for activation of DDR receptors by collagen fibers, although the biological significance of the interaction remains to be determined. In addition, it is puzzling that the docking protein Shc binds to the activated DDR1b isoform, yet MAP kinase is not activated in response to stimulation of DDR receptors by collagen. Finally, the extremely slow kinetics of tyrosine phosphorylation of the DDR receptors in response to collagen stimulation is both unique and unexplained. Perhaps the most intriguing result of the recent observations is that collagen-induced expression of MMP1 is mediated by DDR2 (19Vogel W Gish G.D Alves F Pawson T Mol. Cell. 1997; 1: 13-23Abstract Full Text Full Text PDF PubMed Scopus (750) Google Scholar). It is proposed that overexpression of DDR1 receptors in breast, ovarian, and other epithelial tumors may be responsible for overexpression of MMP1 in the tumor cells which in turn will degrade collagen molecules surrounding the tumor, thus allowing tumor cell escape and metastasis. The validation of this hypothesis in animal models will be of significant interest, and generation of specific inhibitors for DDR1 and DDR2 receptors may prove to be therapeutically beneficial for treatments of metastatic diseases and for scars associated with wound healing.