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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29GM038544-05
Application #
3466306
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1987-07-01
Project End
1993-06-30
Budget Start
1991-07-01
Budget End
1993-06-30
Support Year
5
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Type
Schools of Medicine
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Greenspan, D S; Byers, M G; Eddy, R L et al. (1993) Localization of the human collagen gene COL7A1 to 3p21.3 by fluorescence in situ hybridization. Cytogenet Cell Genet 62:35-6
Lee, S T; Lee, S; Peters, D P et al. (1992) Deletion of the pro-alpha 1(I) N-propeptide affects secretion of type I collagen in Chinese hamster lung cells but not in Mov-13 mouse cells. J Biol Chem 267:24126-33
Greenspan, D S; Byers, M G; Eddy, R L et al. (1992) Human collagen gene COL5A1 maps to the q34.2----q34.3 region of chromosome 9, near the locus for nail-patella syndrome. Genomics 12:836-7
Greenspan, D S; Cheng, W; Hoffman, G G (1991) The pro-alpha 1(V) collagen chain. Complete primary structure, distribution of expression, and comparison with the pro-alpha 1(XI) collagen chain. J Biol Chem 266:24727-33
Greenspan, D S; Lee, S T; Lee, B S et al. (1991) Homology between alpha 2(V) and alpha 1(III) collagen promoters and evidence for negatively acting elements in the alpha 2(V) first intron and 5' flanking sequences. Gene Expr 1:29-39
Lee, S T; Kessler, E; Greenspan, D S (1990) Analysis of site-directed mutations in human pro-alpha 2(I) collagen which block cleavage by the C-proteinase. J Biol Chem 265:21992-6
Greenspan, D S; Hoffman, G G; Lee, B S (1989) High levels of expression of full length human pro-alpha 2(V) collagen cDNA in pro-alpha 2(V)-deficient hamster cells. J Biol Chem 264:20683-7
Lee, S T; Smith, B D; Greenspan, D S (1988) Construction of a full-length cDNA encoding human pro-alpha 2(I) collagen and its expression in pro-alpha 2(I)-deficient W8 rat cells. J Biol Chem 263:13414-8