Identification of Cell Binding Sites in the Laminin 1 Chain Carboxyl-terminal Globular Domain by Systematic Screening of Synthetic Peptides

doi:10.1074/jbc.270.35.20583

Identification of Cell Binding Sites in the Laminin 1 Chain Carboxyl-terminal Globular Domain by Systematic Screening of Synthetic Peptides (*)

From the (1) Laboratory of Developmental Biology, NIDR, National Institutes of Health and the
(2) Laboratory of Medicinal Chemistry, Developmental Therapeutics Program, Division of Cancer Treatment, NCI, National Institutes of Health, Bethesda, Maryland 20892

↵^§ Present address: Dept. of Dermatology, Kobe University School of Medicine, Kobe 650, Japan.
↵^¶ Present address: Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto 606, Japan.

Abstract

The laminin α1 chain carboxyl-terminal globular domain has been identified as a site of multiple biological activities. Using a systematic screening for cell binding sites with 113 overlapping synthetic peptide beads that covered this domain, we found 19 potential active sequences. Corresponding synthetic peptides were evaluated for direct cell attachment, spreading, and inhibition of cell spreading to a laminin-1 substrate using several cell lines. Five peptides (AG-10, AG-22, AG-32, AG-56, and AG-73) showed cell attachment activities with cell-type specificities. Cell spreading on AG-10 was inhibited by β1 and α6 integrin antibodies and on AG-32 was inhibited by β1, α2, and α6 integrin antibodies. In contrast, cell adhesion and spreading on peptide AG-73 were not inhibited by these antibodies. The minimum active sequences of AG-10, AG-32, and AG-73 were determined to be SIYITRF, IAFQRN, and LQVQLSIR, respectively. These sequences are highly conserved among the different species and different laminin α chains, suggesting that they play a critical role for biological function and for interaction with cell surface receptors.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵1 The abbreviations used are:

Fmoc

9-fluorenylmethoxycarbonyl

PBS

phosphate-buffered saline

EMEM

Eagle's minimal essential medium

FBS

fetal bovine serum

BSA

bovine serum albumin

DMEM

Dulbecco's modified Eagle's medium

HEMA

hydroxyethyl methacrylate

mAb

monoclonal antibody.
- Received January 31, 1995.
- Revision received June 1, 1995.