Abstract
Chondroadherin, a leucine-rich repeat family member, contains a very C-terminal sequence CKFPTKRSKKAGRH(359), now shown to bind to heparin with a K(D) of 13 μm. This observation led us to investigate whether chondroadherin interacts via this C-terminal heparin-binding domain with glycosaminoglycan chains of proteoglycans at the cell surface. Cells were shown to bind this heparin-binding peptide in FACS analysis, and the interaction was shown to be with glycosaminoglycans because it was abolished when sulfation was inhibited by chlorate treatment of the cells. In separate experiments, heparin and heparan sulfate inhibited the peptide interaction in a dose-dependent manner. Using a human chondrosarcoma and a murine osteoblast cell line, heparan sulfate proteoglycans were identified as the cell surface receptors involved in the binding. Different binding syndecans were identified in the two different cell lines, indicating that the same protein core of a proteoglycan may have structural and functional differences in the attached heparan sulfate chains. Upon binding to coated peptide, cells spread, demonstrating engagement of the cytoskeleton, but no focal adhesion complex was formed. The number of cells adhering via their β(1) integrin receptor to collagen type II or chondroadherin was profoundly and rapidly enhanced by the addition of the heparin-binding peptide. The peptide added to the cells caused ERK phosphorylation, showing that it triggered intracellular signaling. The results show that heparan sulfate chains differ between various members of the proteoglycan families on a given cell, but also differ between the same proteoglycan on different cells with a potential for differential regulation of cellular activities.
Highlights
Does the C-terminal cationic chondroadherin sequence bind cell surface proteoglycans to modulate cell behavior? Results: Chondroadherin and its C-terminal domain bind tightly to heparin and select proteoglycans, eliciting signals and cell spreading
Cell adhesion to chondroadherin has been shown to be mediated via the integrin ␣21 [23, 24]
The cells do not spread, unlike effects observed when they adhere to other matrix proteins such as collagen or fibronectin
Summary
Does the C-terminal cationic chondroadherin sequence bind cell surface proteoglycans to modulate cell behavior? Results: Chondroadherin and its C-terminal domain bind tightly to heparin and select proteoglycans, eliciting signals and cell spreading. An important element in this regulation is the ability of the cells to use a diversity of surface receptors to interact with matrix proteins or protein fragments These receptors include integrins [8], syndecans [9], and collagens [10], such as those binding to hyaluronan [11, 12]; the discoidin family [11]; as well as receptors for growth factors and cytokines [13]. Heparin-binding Proteins in the Extracellular Matrix integrin-binding and glycosaminoglycan-binding domains, e.g. fibronectin, and the formation of certain signaling complexes depends on targeting more than one cell surface receptor [14]. The isolated chondroadherin C-terminal heparin-binding domain (hbd) of 13 amino acids was shown to stimulate bound chondrocytes to spread and to prominently increase attachment to integrins with the formation of focal adhesion complexes
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