Date of Award
Joan C. Edwards School of Medicine
Type of Degree
Todd L. Green
Leonard J. Deutsch
Laminin, a major component of basement membrane, is a trimeric glycoprotein comprised of three chains - α, β and γ (Burgeson et al., 1994). An order for trimer assembly has been deduced: first, the β and γ chains bind to form a dimer and subsequently α is added to complete the trimer (I. Hunter et al., 1990 & 1992; Utani et al., 1994 & 1995). The C-terminal portions, found within the protein structural domain I of the p and y chains, are implicated in dimer and trimer formation by biochemical studies performed extracellularly (Utani, et al., 1994 & 1995; Nomizu et al., 1995).
Using the yeast two-hybrid system, long arm fragments of the laminin chains β2, γ1, and αl were assayed for their ability to form dimers. This assay confirmed the strong specific interactions between the β and γ chains seen in other studies of recombinant laminin fragments (Nomizu et al., 1994 & 1996; Utani et al., 1994 & 1995). Interactions of the αl fragment with β2 or γ1 were weak or non existent in this assay. A region necessary for dimerization within the β2 chain was found between the C-terminal 75 and 38 amino acids, as the C-terminal 75 amino acids interacted strongly with γl domain I but the C-terminal 38 amino acids did not. Additionally, a domain I fragment of β2 containing a cysteine to serine substitution at amino acid 1765 (created to prevent disulfide bonding) was able to form dimers with y 1 domain I, indicating that noncovalent forces can mediate this interaction.
To determine the ability of domain I alone to mediate specific dimerization of the β2 with the γl chain, the domain I regions of β2 and γl were switched to create two chimeric laminin chains. Epitope-tagged chimeras were tested for their ability to interact with the full-length wild-type β2 or γ1. In immunoprecipitation experiments wild type β2 associated only with the chimera containing domain I of γl and wild-type γ1 coprecipitated only with the chimera containing domain I of β2. These results indicate that the domain I of laminin chains as part of a full-length chain can impart specificity to chain assembly within a cell.
Membrane, Basement – Research.
Molecular biology – Research.
Kamphaus, George D., "The role of domain I in laminin chain assembly" (1998). Theses, Dissertations and Capstones. 1493.