Laminin-binding protein 120 from brain is closely related to the dystrophin-associated glycoprotein, dystroglycan, and binds with high affinity to the major heparin binding domain of laminin.

When brain proteins separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose are probe with 125I-labeled laminin, a single broad band of approximately 120 kDa binds laminin specifically. We show here by two-dimensional electrophoresis and protein microsequencing that this band consists of two distinct laminin-binding proteins. One of these is the amyloid precursor protein. The other, laminin-binding protein (LBP) 120, is closely related to the dystrophin-associated glycoprotein, dystroglycan (156 kDa); 5 peptides from purified bovine brain LBP120, ranging in size from 7 to 19 residues, are up to 100% identical to the predicted amino acid sequence of muscle dystroglycan (ibraghimov-Beskrovanaya, O., Ervasti, J. M., Leveille, C. J., Slaughter, C. A., Sernett, S. W., and Campbell, K. P. (1992) Nature 355, 696-702). These protein microsequence data support the data of Ibraghimov-Beskrovnaya et al., which suggest that the dystroglycan precursor is processed into 120/156- and 43-kDa proteins. Moreover, the data suggest a revision in the position of the proposed cleavage site of the precursor. The glycosylation and extracellular localization of LBP120/dystroglycan are consistent with it being a cell surface laminin receptor. LBP120/dystroglycan, either as a native protein, or following SDS-PAGE and transfer to nitrocellulose, binds with high affinity (Kd = 90 nM) to a proteolytic fragment of laminin (E3) containing the major heparin binding domain. This binding is Ca(2+)-dependent and inhibited by low concentrations of heparin. Thus, LBP120/dystroglycan is a major non-integrin laminin receptor whose high affinity interaction with laminin may reflect a structural role in brain and muscle.

Genetic linkage analysis in recombinant inbred mice of P40, a putative clone for the high-affinity laminin receptor.

Recombinant inbred (RI) mice were used to genetically map sequences of a 43-kDa protein, P40, that was originally identified as a high-affinity laminin receptor. More recent data have implicated this protein in development of the retina (Rabacchi et al. Development 109: 521-531, 1990), possibly via its proposed function in protein translation (G. Brawerman, personal communication). We have identified, in Southern blots, a set of P40-related sequences in BXD recombinant inbred mouse DNA. Ten restriction fragment length polymorphisms (RFLPs) segregate among the RI strains and display strain distribution patterns (SDPs) which are linked in varying degrees to previously typed loci on Chromosomes (Chrs) 1, 3, 6, 9, 11, 14, and 19. An intronic DNA probe from an incompletely processed P40 mRNA transcript overlaps with two of these loci mapping near the cholecystokinin gene locus on the distal arm of Chr 9 and to another site on the distal arm of Chr 6, suggesting that functional genes probably reside at least at these two sites. These P40 loci comprise part of a multimember gene family that is well dispersed in the mouse genome.

Isolation and partial characterization of high affinity laminin receptors in neural cells.

Neural cells in culture (NG-108, PC12, chick dorsal root ganglion, chick spinal cord, and rat astrocytes) bind laminin with an apparent Kd of congruent to 10(-9) M. Laminin affinity chromatography of chick brain membranes washed with 150 mM NaCl and eluted with 0.2 M glycine buffer, pH 3.5, yields a single protein with an apparent molecular mass of 67 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Isoelectric focusing and peptide mapping indicate that the 67-kDa protein is distinct from bovine serum albumin (68 kDa) but indistinguishable from high affinity laminin receptors isolated from skeletal muscle. After electroblotting onto nitrocellulose paper and probing with 125I-laminin, this putative laminin receptor binds laminin specifically (100 ng/ml). A second protein (congruent to 120-140 kDa) is also detected with 125I-laminin (100 ng/ml) in the laminin affinity-purified membrane proteins. Both 67- and congruent to 120-140-kDa proteins can be laminin affinity-purified from cultures enriched for neurons (greater than 90%) following metabolic labeling with [35S]methionine. Our data suggest that neural cells (dorsal root ganglion, central nervous system neurons, astrocytes, and several neural cell lines) have high affinity binding sites for laminin and that two membrane proteins, 67- and congruent to 120-140-kDa, are responsible at least in part for this binding.