This proposal examines the hypothese that type I and III procollagen molecules contain transport signals and/or binding determinants that govern: 1) the molecules' movement from rough endoplasmic reticulm (RER), to Golgi, to cell surface; and 2) the incorporation of procollagen into the extracellular matrix (ECM). The experimental approach is to transfect eukaryotic cells with normal and mutagenized human type I and III DNAs and to then analyze transcription, synthesis, transcellular transport, secretion and martix production in the in vitro transient and long- term expression systems. These studies will require: a) the construction of complete cDNAs for proalphs1 (I) and proalpha1 (III) by probing a special cDNA fibroblast library enriched for cDNAs representative of the 5' and middle portions of collagen genes, and then cutting and splicting the recovered cDNA segments as necessary; b) introduction of selected mutations (deletions, insertions, base replacements) into the cDNAs for proalpha1 (I), proalpha2 (I) and proalpha1 (III). This will involve identifying useful restriction sites for excisions and insertions, and oligonucleotide copying for site-directed mutagenesis; c) insertion of the cDNAs into efficient expression vectors; and d) evaluation of PC12 cells, HeLa cells, COS cells, cells of lymphocytic lineage, and certain useful mutant mouse and human fibroblast lines and strains, as transfection recipients for the various analyses. In this manner, we hope to define domains within the procollagen molecules that govern transport, secretion and fibrillogenesis. We anticipate that the complete cDNAs and transfection systems developed in this program will be useful to investigators examining other aspects of collagen biology. These studies should increase our understanding of protein secretion in general, and procollagen secretion in particular. Given the central role of collagen deposition in morphogenesis and wound repair, these studies should impact on many biomedical problems.
