Molecular analysis of laminin N-terminal domains mediating self-interactions

Abstract

The ability of laminins to self-polymerize is crucial for the formation of basement membranes. Development of this polymerized network has profound effects upon tissue architecture as well as on the intracellular organization and differentiation of neighboring cells. The laminin N-terminal (LN) domains have been shown to mediate this interaction and studies using proteolytic fragments derived from laminin-1 led to the theory that network assembly depends on the formation of a heterotrimeric complex between LN domains derived from α, β, and γ chains in different laminin molecules with homologous interactions being insignificant. The laminin family consists of 15 known isoforms formed from five α, three β, and three γ chains, of which some are truncated and lack the N-terminal LN domain. To address whether the model of heterotrimeric complex formation is applicable to laminin isoforms other than laminin-1, eight LN domains found in the laminin protein family were recombinantly expressed and tested in three different assays for homologous and heterologous interactions. The results showed that the lack of homologous interactions is an exception, with such interactions being seen for LN domains derived from all α chains and from the β₂ and β₃ subunits. The γ chain-derived LN domains showed a far more limited binding repertoire, particularly in the case of the γ₃ chain, which is found present in a range of non-basement membrane locations. Further, whereas the interactions depended upon Ca²⁺ ions, with EDTA reversibly abrogating binding, EDTA-induced conformational changes were not reversible. Together these results demonstrate that the assembly model proposed on the basis of laminin-1 may be a simplification, with the assembly of naturally occurring laminin networks being far more complex and highly dependent upon which laminin isoforms are present.

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