The objective of this proposal is to investigate the mechanism and consequences of cell adhesion to a novel cell adhesion protein - matrix Gla protein (MGP). the long-term goal of these studies is to provide a more complete understanding of the interactions between cells and the extracellular matrix with specific emphasis on cartilage and bone. The study of cell adhesion to MGP is of particular interest since the protein contains the unique amino acid, gamma-carboxyglutamic acid (Gla), which is produced in a posttranslational gamma-carboxylation reaction requiring reduced vitamin K as cofactor. MGP is the first cell adhesion protein known to have this modification. MGP is found primarily in cartilage and bone, two tissues where loss of the extracellular matrix results in two very prevalent, chronic, disabling diseases, osteoarthritis and osteoporosis. The synthesis of gamma-carboxylated MGP appears to be necessary for normal development of growth plate cartilage. Overexpression of MGP has been noted in a breast cancer cell line and in primary tumors from prostate, testicular and renal cancers. Increased MGP has also been noted in osteoarthritic cartilage. Therefore, an investigation of cell adhesion to MGP will provide important information about cartilage and bone metabolism and potentially new insight into tumor growth and metastasis. To accomplish the objectives of this proposal specific aims are focused to: 1) determine the mechanism by which cells bind to MGP by elucidating the MGP cell-binding domain. The binding domain will be identified by competition experiments in a cell adhesion assay using peptide fragments derived from MGP by proteolysis. The cell receptor for MGP will be isolated and identified using ligand affinity chromatography as the key step for isolation; 2) determine the cell-type specificity for adherence to MGP including an investigation of tumor cell adherence to MGP, correlate adherence with MGP expression and synthesis, and compare adhesion to MGP with other cartilage and bone proteins; 3) determine if MGP provides a link between the cell and other extracellular matrix proteins by investigating the relationship of MGP to the other matrix proteins in colocalization studies using electron microscopy and by studies where protein to protein interactions are investigated by affinity techniques; 4) determine if cell adhesion to MGP has an effect on cell function by studying collagen and proteoglycan synthesis by pre- chondrocytes (mesenchymal cells), dedifferentiated chondrocytes, and chondrocytes that have attached to MGP and; 5) determine the chronological appearance and localization of MGP in developing bone and cartilage using immunocytochemical and in-situ hybridization techniques. This work should provide new insight into the function of one of the extrahepatic vitamin K-dependent proteins and unveil a novel mechanism of cell adhesion which couples cell adhesion to vitamin K-function and metabolism in cartilage and bone.
