ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 290, No. 2, November 1, pp. 320-325,1991 Identification of a 1 IO-kDa Nonintegrin Cell Surface Laminin-Binding Protein Which Recognizes an A Chain Neurite-Promoting Peptide Hynda K. Kleinman,*,’ Benjamin S. Weeks,* Frances B. Cannon,* Thomas M. Sweeney,? Gregory C. Sephel,$ Bruno Clement,§ Mona Zain,* Mark 0. J. Olson,lI Mathias Jucker,” and Beth A. Burrous** *Laboratory of Developmental Biology, National Institute of Dental Research, NIH, Bethesda, Maryland 20892; tP.0. Box 5626, Charlottesville, Virginia 22905; $Vanderbilt University School of Medicine, Laboratory Service, 1310 24th Avenue, No. 5, Nashville, Tennessee 37203; §Inserm U-49, Hospital Pontchaillou, 35033 Rennes Ceder, France; IDepartment of Biochemistry, University of Mississippi Medical Center, Jackson, Mississippi 39216-4505; 11 Laboratory of Cellular and Molecular Biology, Institute on Aging, NIH, Baltimore, Maryland 21224; **U.S. Patent and Trademark Oflice, Arlington, Virginia 22202 Received April 25, 1991, and in revised form June 24, 1991 Laminin is a potent promoter of neurite outgrowth, and a synthetic peptide of 19 amino acids, PA22-2, from the A chain has been found to promote process formation. Using peptide affinity chromatography, we have identi- fied a 1 lo-kDa, cell surface ligand from both neural cells and brain which binds this sequence. This binding protein does not share immunological identity with the B 1 chain of integrin, and reduction does not alter its mobility in sodium dodecyl sulfate-polyacrylamide gel electropho- resis. Antibody to the 1 IO-kDa protein stained cellular processes in vivo. Sequence analysis of the first 18 amino acids from the amino terminus yielded almost exact se- quence identity with nucleolin, a major llO-kDa nu- cleolar phosphoprotein. Antibody to nucleolin, however, does not interact with the neural-derived, laminin-pep- tide-binding 1 lo-kDa protein. The 1 IO-kDa protein ap- pears to be a ligand for a specific site on laminin. o 1991 Academic Press, Inc. Laminin, a major glycoprotein of basement membranes, has many biological activities with various cultured cells (l-4). The three chains of laminin designated A(400 kDa), Bl(210 kDa), and B2(200 kDa) are held together in a cross-like structure (5-7). Five active sites for cell adhe- sion have been identified using synthetic peptides, and include YIGSR (residues 929-933 on the Bl chain), PDSGR (residues 902-906 on the Bl chain), CQFALRG- ’ To whom correspondence should be addressed at NIH, NIDR, Bldg. 30, Rm. 407, Bethesda, MD 20892. 320 DNP (residues 1115-1124 on the A chain), F9 (residues 641-660 on the Bl chain), and PA22-2 CSRARKQAAS- IKVAVSADR (residues 2091-2108 on the A chain) (8- 13). In addition to promoting cell adhesion, the first three inhibit in vim experimental metastasesof murine Bl6FlO melanoma cells. YIGSR-NH2 is the most active in re- ducing tumor cell colonization of the lungs (14,15). PA22- 2 promotes cell spreading, neurite outgrowth, and B16FlO melanoma colonization of the lungs (16,17). PA22-2 also increases collagenase production by various cells. These activities mimic whole laminin but are distinct from those observed with other active synthetic peptides. Several cellular receptors have been described for lam- inin including integrins (crl, (~2, a3, and a6), a 32-/67- kDa laminin-binding protein, sulfatides, a 35kDa lectin, galactosyl transferase, and a llO-kDa protein (18-31). Nothing is known about the cell surface ligands for the peptides F9 and PDSGR. The A chain CQFALRGDNP site sequence contains an RGD which is known to be an integrin-binding site on various adhesion proteins, in- cluding fibronectin, vitronectin, collagen, etc. (25). The YIGSR sequence has been shown to interact with the 32- /67-kDa laminin-binding protein (8). Using peptide af- finity chromatography, we describe here the isolation and characterization of a llO-kDa cell-binding protein, which recognizes the PA22-2 sequence. A llO-kDa laminin- binding protein has been previously described in neuronal (26, 28, 29) and Sertoli cells (32). A partial amino acid sequence of the brain-derived llO-kDa protein closely matches a phosphorylated nucleolar protein, nucleolin, which is involved in ribosome biogenesis (33-35) but the two molecules are likely distinct. 0003.9861/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form resewed.