Basement membranes are thin sheets of extracellular matrix that separate dissimilar cells and tissues. This matrix is difficult to isolate from native tissues but tumor cell lines which exclusively produce basement membrane have proven useful as a model system for identifying and characterizing the major components of this type of extracellular matrix. A heparan sulfate containing proteoglycan has been isolated and partially characterized using these model cell lines. Its core protein is an exceptionally large gene product (400,00 Mr) containing homologies to N- cam, laminin and low density lipoprotein receptor in the regions os far cloned. This proteoglycan, or its counterpart in native and cell attachment. The long term objective of this proposal is to establish the complete structure of the proteoglycan and identify the process involved in the regulation of its production.
The specific aims are: (1) to complete the characterization of the proteoglycan core protein gene product by constructing cDNA libraries and, using existing clones, isolate the cDNA clones needed to deduce the entire core protein structure; (2) to characterize the proteoglycan gene by using the cDNA clones to isolate genomic clones, to determine the intron/exon structure and to identify the cis-acting region of the gene which controls transcription; (3) to establish the regulation of proteoglycan gene expression during differentiation of F9 cells by using the cDNA clones to measure the kinetics of change of mRNA levels and mRNA synthesis and mRNA levels upon response to exogenous matrix. The results of these studies will provide an understanding of the proteoglycan core protein structure and the mechanisms involved in its production during the biogenesis and maintenance of basement membranes.
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