Basement membranes are thin sheets of extracellular matrix that separate endothelial, epithelial and muscle cells, as well as the nervous system, from adjacent cells and connective tissues. All basement membranes are composed primarily of collagen IV, laminin, entactin and perlecan, a large heparan sulfate containing proteoglycan. These components interact to provide the fundamental architectural framework of basement membranes. Perlecan is a critical participant in a number of diverse biological functions such as ionic filtration, cell-attachment and matrix assembly that are needed for the development and maintenance of tissues and organs. Perlecan's 396 kDa core protein consists of five major domains; one with a potential site for heparan sulfate attachment and the remaining four showing homology to LDL receptor, laminin, N-CAM and agrin. The long term goals of this project are to determine the specific roles of the different domains in heparan sulfate synthesis, cell adhesion, matrix interaction and embryonic development. We will do this by using the cDNA clones to perlecan to express individual domains, selected groups of domains and full length perlecan as recombinant proteins and then evaluating their activities and properties.
The first aim i s to identify the domain that initiates heparan sulfate synthesis and the domains that influence this activity by biosynthetically radiolabeling cells producing the recombinant proteins and measuring the amount of heparan sulfate on the protein.
The second aim i s to identify the domains that regulate cell adhesion by purifying the recombinant proteins, coating the proteins on dishes and measuring the number and type of cells that attach to the dish.
The third aim i s to identify the domains that interact with collagen IV, laminin and entactin by mixing the purified radiolabeled recombinant proteins with each of these components, capturing the complex with an antibody and measuring the amount of protein in the complex.
The fourth aim i s to identify new biological functions for perlecan by producing a perlecan dominant negative mutation in mice and evaluating its consequences on growth and development. The information obtained from these studies will identify the biological functions of each domain of perlecan and provide new insights into the role of perlecan in growth and development.
| Smith, Simone M-L; West, Leigh A; Hassell, John R (2007) The core protein of growth plate perlecan binds FGF-18 and alters its mitogenic effect on chondrocytes. Arch Biochem Biophys 468:244-51 |
| Dolan, M; Horchar, T; Rigatti, B et al. (1997) Identification of sites in domain I of perlecan that regulate heparan sulfate synthesis. J Biol Chem 272:4316-22 |
| Chakravarti, S; Phillips, S L; Hassell, J R (1991) Assignment of the perlecan (heparan sulfate proteoglycan) gene to mouse chromosome 4. Mamm Genome 1:270-2 |
