The general aim of the proposal is to define the mechanisms that govern the secretion of procollagen and its conversion to fibrillar forms in the extracellular matrix. Such knowledge is critical for an understanding of matrix-cell interactions in the course of embryonic differentiation and tissue repair after injury. The major hypotheses of the proposal are: 1) that native, soluble type I procollagen is bound at the fibroblast surface in the course of its secretion; 2) that the procollagen binds to a unique receptor that may also function in the transcellular transport of procollagen; and 3) that surface binding of procollagen is determinate for fibrillogenesis and required for incorporation of procollagen into fibrils in the extracellular matrix (ECM). Experiments include: 1) ruling out fibronectin or fibronectin cell receptors as determinants of procollagen binding; 2) following the movement of added 125I-procollagen ligand and metabolically-labeled procollagen from cell surface to insoluble ECM in short (0-3 hr.) and long (0-25 hr.) labeling and chase experiments; 3) assessing the role of disulfide multimer assembly for incorporation of procollagen into the ECM; 4) identifying the intermediates of procollagen processing that are cell-surface bound and that are incorporated into the ECM; 5) characterizing the procollagen receptor protein(s) in the plasma, smooth and ERR membranes by means of nitrocellulose filter binding assays, affinity chromatography, PAGE, western blotting with 125I-procollagen as the probe, and labeling the receptor(s) with photoaffinity cross-linkers; 6) developing polyclonal and monoclonal antibodies to the receptor(s) and employing them in the studies of receptor structure, function, localization and metabolism; and 7) using gold-labeled antibodies to procollagen to visualize at the ultrastructural level specific binding of exogenously added procollagen to the fibroblast surface.
| Dzamba, B J; Wu, H; Jaenisch, R et al. (1993) Fibronectin binding site in type I collagen regulates fibronectin fibril formation. J Cell Biol 121:1165-72 |
| Dzamba, B J; Peters, D M (1991) Arrangement of cellular fibronectin in noncollagenous fibrils in human fibroblast cultures. J Cell Sci 100 ( Pt 3):605-12 |
