Paxillin Binding to a Conserved Sequence Motif in the α4 Integrin Cytoplasmic Domain

doi:10.1074/jbc.M000388200

Paxillin Binding to a Conserved Sequence Motif in the α₄ Integrin Cytoplasmic Domain*

Shouchun Liu‡ and
Mark H. Ginsberg§

From the Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037

Abstract

α₄β₁integrin-mediated cell adhesion results in increased cell migration, reduced cell spreading, and focal adhesion formation relative to other β₁ integrins. Paxillin, a signaling adapter protein, binds tightly to the α₄ cytoplasmic domain and is implicated in α₄ integrin signaling. We now report the mapping of a paxillin-binding site in the α₄ cytoplasmic domain and an assessment of its role in the α₄tail-specific integrin functions. By using truncation mutants and a peptide competition assay, we found that a region of 9 amino acid residues (Glu⁹⁸³-Tyr⁹⁹¹) within the α₄ cytoplasmic domain contains a minimal sequence sufficient for paxillin binding. Alanine scanning of this region implicated Tyr⁹⁹¹ and Glu⁹⁸³ as critical residues. The role of these residues was confirmed by introducing these Ala substitutions into the full-length α₄ tail sequence. Y991A or E983A substitution disrupted the interaction of α₄ integrins with paxillin. These same two point mutations reversed the effects of the α₄ tail on cell spreading. The key features of the identified paxillin-binding sequence are present in all α₄ integrins sequenced to date, including that from Xenopus laevis. The maintenance of this sequence motif suggests that paxillin binding is an evolutionarily conserved function of α₄ integrins.

Footnotes

↵* This work was supported in part by grants from the National Institutes of Health. This is publication number 13010VB from the Scripps Research Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ Supported by National Service Research Award IF32HL09922-01.
↵§ To whom correspondence should be addressed: Dept. of Vascular Biology, VB-2, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-784-7143; Fax: 858-784-7343; E-mail: ginsberg@scripps.edu.
Published, JBC Papers in Press, April 25, 2000, DOI 10.1074/jbc.M000388200
↵2 S. Liu and M. H. Ginsberg, unpublished data.
Abbreviations:

CHO cells

Chinese hamster ovary cells

GST

glutathione S-transferase

BSA

bovine serum albumin

Fg

fibrinogen

FN

fibronectin

Pipes

1,4-piperazinediethanesulfonic acid

PMSF

phenylmethylsulfonyl fluoride

PAGE

polyacrylamide gel electrophoresis

PBS

phosphate-buffered saline

FAK

focal adhesion kinase
- Received January 19, 2000.
- Revision received April 14, 2000.
The American Society for Biochemistry and Molecular Biology, Inc.