Laminins are the key proteins for proper basement membrane assembly
Basement membrane composition is highly cell-surface selective and for proper assembly, laminins are the key proteins. Laminin molecules self-assemble via a thermodynamically unfavorable nucleation binding followed by a calcium-dependent polymerization of the LN domains in the short-arms of the α, β, and γ chains (Yurchenco et al., 1985; Carafoli et al., 2012; Yurchenco & Cheng, 1993; Purvis & Hohenester, 2012). The sheet-like laminin network binds to other proteins in the basement membrane. Laminin interact with nidogen via LE motifs of the γ1 and γ3 chains (Gersdorff et al., 2005; Takagi et al., 2003; Stetefeld et al., 1996), and the Lβ domain of the β chains binds to agrin (Domogatskaya et al., 2012). Laminin is linked to collagen IV through nidogen and heparin interactions, forming a covalently stabilized network (Hohenester & Yurchenco, 2013).
Laminin cell membrane receptor interactions
The laminin network formation is facilitated by cooperatively cell surface binding via the domains in the C-terminal end (Yurchenco, 2015). There are many binding sites on the full-length laminin molecule which can interact with cell membrane receptors, in particular integrins, α-dystroglycan (αDG), and sulfated glycolipids (SGLs).
Integrins are important laminin receptors that initiates intracellular signaling pathways. Integrins are heterodimeric transmembrane membrane proteins consisting of an α and an β subunit. There has been at least 24 distinct integrins identified and the type and quantity of integrins on each cell are cell- and tissue-specific (Campbell & Humphries, 2011). The α5-laminins (especially LN-511) exhibit the broadest degree of integrin binding interactions (Miner et al., 1995). In contrast, the α2-laminins interact only with the α7β1 integrin and dystroglycan. The integrin α6 chain forms the major receptors for ECM laminins and integrin α6β1 binds to most laminin isoforms (Colognato & Yurchenco, 2000).
The molecular mechanism underlying the laminin-mediated signaling to integrins and other signaling receptors is still not clear. Studies indicate that the LG domains in C-terminal region of laminins are highly involved in laminin recognition by integrins and that the integrins that bind to laminin LG domains also require that the coiled coil be present (Deutzmann et al., 1990; Sung et al., 1993). A short C-terminal γ1 segment that contain a critical glutamic acid that extends out beyond the coiled-coil have also shown to play an important role (Ido et al., 2007). αDG binds primarily to the LG4 C-terminal domain and, to a lesser degree, to the LG1-3 domains of the laminins. SGLs bind to the LG4 and LG5 domains (Wizemann et al., 2003; Yu & Talts, 2003; Aumailley et al., 2005). Integrins and SGLs also interact with some of the N-terminal globular domains (Aumailley et al., 2005; Yurchenco, 2011). LG4 and N-terminal LE domains also binds to sydecans which regulates cell adhesion and motility (Okamoto et al., 2003; Ogawa et al. 2007). Beside direct interaction, laminin may also link to cell surface α-DG through secreted protein agrin (Denzer et al., 1998) or perlecan (Talts et al., 1999).
Only the intact, full length laminin molecules can replicate a biological relevant milieu for the cells
The expression of laminin isoforms in the basement membrane is heterogeneous and tissue specific. Without the right combination of laminin isoforms cells and tissues becomes dysfunctional. Laminin binding to cell membrane receptors elicit both mechanical and chemical signals in the cell, regulating organization of the cytoskeleton, cell adhesion, motility, proliferation and differentiation. Laminin-laminin linkage induces individual laminin affinities to surface binding molecules and the changes in the laminin polymer stiffness may alter the signaling strength (Yurchenco, 2015). Laminins are capable of co-signaling with growth factors and efficiently buffers endogenously produced growth factors (Rodin et al., 2014), which adds to the mechanistic complexity. Cell polarization generated by laminin interactions can also affect the cellular response to treatments with growth factor (Domogatskaya et al., 2012).
A fractionated or truncated laminin molecule or laminins isolated from tissue, lack many of the laminin domains which is needed by the cells for the proper extracellular network to form and to for stimulation of correct cellular signal transductions. Hence, only the intact, full length laminin is able to replicate the full biological relevance. Emphasizing on biology by mimicking the natural matrix protein composition is one of the most important aspects to achieve phenotypically relevant, stable cell cultures and reproducible protocols.
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