Identification and characterization of the integrin alpha2beta1 binding motif in chondroadherin mediating cell attachment.
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.
Department of Clinical Sciences Lund, Lund University, BMC Plan C12, SE-22184 Lund, Sweden.
Lisbet
L
Haglund
Viveka
V
Tillgren
Laura
L
Addis
Christina
C
Wenglén
Anneliese
A
Recklies
Dick
D
Heinegård
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002417:Cattle; D002448:Cell Adhesion / Q000187:drug effects; D002455:Cell Division; D048430:Cell Shape; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D038982:Integrin alpha2beta1 / Q000378:metabolism* / Q000502:physiology; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D010456:Peptides, Cyclic / Q000138:chemical synthesis / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology
D016326:Extracellular Matrix Proteins; D038982:Integrin alpha2beta1; D010456:Peptides, Cyclic; C088980:chondroadherin; D048049:Extracellular Signal-Regulated MAP Kinases; D020929:Mitogen-Activated Protein Kinase Kinases