Identification and Characterization of the Integrin α2β1 Binding Motif in Chondroadherin Mediating Cell Attachment

Lisbet Haglund,Viveka Tillgren,Laura Addis,Christina Wenglén,Anneliese Recklies,Dick Heinegård

doi:10.1074/jbc.m110.161141

Abstract

Chondroadherin is a leucine-rich repeat protein known to mediate adhesion of isolated cells via the integrin α(2)β(1) and to interact with collagen. In this work, we show that cell adhesion to chondroadherin leads to activation of MAPKs but does not result in cell spreading and division. This is in contrast to the spreading and dividing of cells grown on collagen, although the binding is mediated via the same α(2)β(1) receptor. We identified a cell binding motif, CQLRGLRRWLEAK(318) by mass spectrometry after protease digestion of chondroadherin. Cells adhering to the synthetic peptide CQLRGLRRWLEAK(318) remained round, as was observed when they bound to the intact protein. The peptide added in solution was able to inhibit cell adhesion to the intact protein in a dose-dependent manner and was also verified to bind to the α(2)β(1) integrin. A cyclic peptide, CQLRGLRRWLEAKASRPDATC(326), mimicking the structural constraints of this sequence in the intact protein, showed similar efficiency in inhibiting binding to chondroadherin. The unique peptide motif responsible for cellular binding is primarily located in the octamer sequence LRRWLEAK(318). Binding of cells to the active peptide or to chondroadherin immobilized on cell culture plates rapidly induces intracellular signaling (i.e. ERK phosphorylation). Thus, chondroadherin interaction with cells may be central for maintaining the adult chondrocyte phenotype and cartilage homeostasis. The peptides, particularly the more stable cyclic peptide, open new opportunities to modulate cell behavior in situations of tissue pathology.

Highlights

Cartilage contains one specific type of cell, the chondrocyte, surrounded by an extensive extracellular matrix accounting for ϳ90% of the tissue volume
We found that adhesion to matrilin-1 decreased after collagenase treatment to background levels seen with BSA (Fig. 1), indicating that cell surface collagens mediate adhesion to matrilin-1
Binding of cells to chondroadherin was partially decreased upon collagenase treatment (Fig. 1), suggesting that this molecule may bind to cell surface collagens in addition to integrins

Summary

Introduction

Cartilage contains one specific type of cell, the chondrocyte, surrounded by an extensive extracellular matrix accounting for ϳ90% of the tissue volume. Cells orchestrate and regulate the formation and maintenance of the extracellular matrix by interacting with their surroundings via a number of different receptors, such as integrins [4], heparan sulfate proteoglycans [5], hyaluronan receptors [6], and discoidin receptors [7]. In the case of fibronectin, treatment with a heparin-binding fragment of the molecule (HepII), a peptide from this domain, or phorbol esters to activate protein kinase C is necessary to provide sufficient signals inducing spreading, formation of focal adhesions, and stress fibers. This process involves both integrin and syndecan receptors at the cell surface. Identification of a specific peptide mediating the binding may provide an opening for the design of compounds promoting cartilage repair

Methods

Results

Conclusion