Identification of the Binding Site for the Lutheran Blood Group Glycoprotein on Laminin α5 through Expression of Chimeric Laminin Chains in Vivo

Abstract

The Lutheran blood group glycoprotein (Lu), also known as basal cell adhesion molecule, is an Ig superfamily transmembrane receptor for laminin ␣5. Lu is expressed on the surface of a subset of muscle and epithelial cells in diverse tissues and is thought to be involved in both normal and disease processes, including sickle cell disease and cancer. Here we investigated the binding of Lu to laminin ␣5 in vivo and in vitro. We prepared a soluble recombinant Lu (sol-Lu) composed of the Lu extracellular domain and a His 6 tag. Sol-Lu bound specifically to laminin-10/11 (␣5␤1/␤2␥1) in enzyme-linked immunosorbent assays and bound to bona fide basement membranes containing laminin ␣5 in tissue sections. Sol-Lu did not bind to tissue sections of laminin ␣5 knockout embryos, despite the fact that the four other ␣ chains were present. To identify the Lubinding site on laminin ␣5, we prepared modified ␣5 cDNAs encoding chimeric laminins containing all or part of the laminin ␣1 G domain in place of the analogous ␣5 regions. These constructs were used to generate transgenic mice. Proteins derived from transgenes were detected in basement membranes and were assayed for their ability to bind Lu by examining the localization of endogenous Lu and the binding of sol-Lu applied to tissue sections. Our results demonstrate that the ␣5 LG3 module is essential for Lu binding to laminin ␣5.

Identification of an Active Site on the Laminin α5 Chain Globular Domain That Binds to CD44 and Inhibits Malignancy

“…Laminin-10/11 is recognized by several different receptors, including integrins ␣3␤1, ␣6␤1, ␣6␤4, Lutheran blood group glycoprotein, and dystroglycan (22)(23)(24). Furthermore, human colon carcinoma cell migration on laminin-10 is mediated by integrins ␣3␤1, ␣6␤4 (25).…”

Partial Rescue of Glomerular Laminin α5 Mutations by Wild-Type Endothelia Produce Hybrid Glomeruli

“…consists of five homologous structural subunits (LG1 through 5) 22 and mediates binding to integrins, dystroglycan, Lutheran, and/or other laminin receptors on a variety of cells. 23,24 For examination of LG function in detail, transgenic mice in which all or part of the ␣5 LG domain was replaced with corresponding regions of the human ␣1 LG domain were created. 25 These laminin variants were then mated onto the laminin ␣5 knockout background.…”

Basement Membranes: Cell Scaffoldings and Signaling Platforms

“…Failure of the laminin b1 to b2 transition was found to result in a normalappearing GBM associated with proteinuria whereas failure of replacement of laminin a1 by a5 was found to result in a failure of the glomerular tuft to develop within Bowman's space and a failure of normal organization of the glomerular cell types, effects likely caused by absence of a5-laminin receptor interactions (Noakes et al 1995b;Miner and Li 2000;Moulson et al 2001). The unique receptorbinding activities within the laminin a5 subunit, not found in the laminin a1 compensating subunit and required for glomerular vascularization, were localized to the LG1-3 domains that mediate integrin a3b1 and Lutheran receptor binding (Kikkawa et al 2002;Kikkawa and Miner 2006).…”