Date of Award

1998

Degree Name

Biomedical Sciences

College

Joan C. Edwards School of Medicine

Type of Degree

Ph.D.

Document Type

Thesis

First Advisor

Stephen Fish

Second Advisor

William McCumbee

Third Advisor

Donald Primerano

Fourth Advisor

Vernon Reichenbecher

Fifth Advisor

Todd L. Green

Sixth Advisor

Leonard J. Deutsch

Abstract

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.

Subject(s)

Membrane, Basement – Research.

Molecular biology – Research.

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