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 ␣, ␤, and ␥ chains in different laminin molecules with homologous interactions being insignificant. The laminin family consists of 15 known isoforms formed from five ␣, three ␤, and three ␥ 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 ␣ chains and from the ␤ 2 and ␤ 3 subunits. The ␥ chain-derived LN domains showed a far more limited binding repertoire, particularly in the case of the ␥ 3 chain, which is found present in a range of non-basement membrane locations. Further, whereas the interactions depended upon Ca 2؉ 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.Basement membranes are specialized extracellular matrices found underlying all epithelia and endothelia as well as surrounding many types of mesenchymal cells. Laminins constitute the major noncollagenous protein component within the basement membrane and are crucial for its formation (1, 2). Through their interactions with specific receptors, especially members of the ␤ 1 integrin family and ␣-dystroglycan, they induce many cellular effects, including differentiation and cellular and axonal migration. The prototype, laminin-1, isolated from embryonic carcinoma cells (3) or the Engelbreth-HolmSwarm tumor (4) has been shown to belong to a family consisting of 15 members (for nomenclature, see Refs. 5-7). Laminins are multidomain heterotrimers formed by the combination of one ␣, one ␤, and one ␥ chain. Laminin-1 is an 800-kDa glycoprotein composed of a 400-kDa ␣ 1 chain and ␤ 1 and ␥ 1 chains, each of 200 kDa. It has a cross structure with one long and three short arms, the latter being formed from the three free N-terminal ends of the ␣, ␤, and ␥ chains (8). These parts of the ␤ and ␥ chains each contain two globular domains, designated IV and VI, whereas there are three...

Binding of Netrin-4 to Laminin Short Arms Regulates Basement Membrane Assembly

“…As shown previously and in this cross-linking experiment, the N-terminal ⌬laminin ␥1 chain fragments do not self-interact (Fig. 5A) (37). ⌬Netrin-4 on the other hand shows self-interaction as well as an interaction with ⌬laminin ␥1 chain fragments FIGURE 2.…”

“…Some laminin LN domains undergo a conformational change in the presence of Ca 2ϩ (37). To determine whether the conformation of ⌬netrin-4 is also influenced by Ca 2ϩ , circular dichro- ELISA style ligand binding assays were performed using native laminin-111 (trimer consisting of the laminin chains ␣1, ␤1, and ␥1 and isolated from mouse EHS tumor).…”

Basement Membranes: Cell Scaffoldings and Signaling Platforms

“…3A). Examination of binding among recombinant LN/LEa fragment pairs confirmed the predicted pairing of a1LN-b1LN, a1LN-g1LN, and b1LN-g1LN and absence of b1LN and g1LN self-binding (Odenthal et al 2004). The analysis also revealed self-aLN binding, interaLN binding, inter-b2LN binding, an absence of g3LN interactions with LN/LE domains other than those of b2 and b3, an absence of a2LN-g1LN binding, and a range of LN-LN affinities, all implying greater complexity of selfassembly, particularly among the laminin isoforms.…”

laminin alpha 1gene is essential for normal lens development in zebrafish

“…The highest level of conservation was identified in the N-terminal domain of lama1 followed by the laminin globular (G-like) domains. The N-terminal domain was shown to be involved in laminin polymerization in vitro [41,42], and binding to integrins α1β1 and α1β2 [43]. The five laminin globular domains located in the C-terminus represent the main cell-adhesive sites and bind the major laminin receptor integrin α6β1 as well as α6β4 and α7β1 [44], extracellular heparan proteoglycan perlecan, dystroglycan, sulfatides and heparin [45]; mice lacking the alpha 1 chain LG4-5 module were reported to die at E6.5 with failure of epiblast differentiation [46].…”