A Conserved Sequence Motif in the Integrin β3 Cytoplasmic Domain Is Required for Its Specific Interaction with β3-Endonexin

doi:10.1074/jbc.272.12.7693

A Conserved Sequence Motif in the Integrin β₃ Cytoplasmic Domain Is Required for Its Specific Interaction with β₃-Endonexin*

From the Departments of ^‡ Vascular Biology, and
^∥ Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037

** To whom correspondence should be addressed:
Dept. of Vascular Biology, Scripps Research Inst., 10550 North Torrey Pines Rd., La Jolla, CA 92037.
Tel.: 619-784-7118; Fax: 619-784-7343; E-mail: ginsberg{at}scripps.edu

Abstract

Integrin signaling is mediated by interaction of integrin cytoplasmic domains with intracellular signaling molecules. Recently, we identified a novel 111-amino acid polypeptide, termed β₃-endonexin, which interacts selectively with the integrin β₃ cytoplasmic domain. In the present study we conducted a systematic mutational analysis of both the integrin β₃ cytoplasmic domain and β₃-endonexin to map sites required for interaction. The interaction of the full-length β₃ integrin subunit with β₃-endonexin in vitro required the β₃ cytoplasmic domain. In a yeast two-hybrid system, both membrane-proximal and membrane-distal residues of the β₃ cytoplasmic domain were necessary for interaction with β₃-endonexin. In particular, the membrane-distal NITY motif at β₃ 756-759 was critical for the interaction. Exchange of β₃ residues 756-759 (NITY) for the corresponding residues in β₁ (NPKY) endowed the β₁ cytoplasmic domain with the ability to interact with β₃-endonexin. Conversely, exchange of the NPKY motif at β₁ 772-775 for the NITY motif in β₃ abolished interaction of this chimeric cytoplasmic domain with β₃-endonexin. Because the NITY motif is present in the β₃ but not the β₁ cytoplasmic domain, these results explain the selective interaction of this cytoplasmic domain with β₃-endonexin. In addition, deletional analysis suggested that a core 91-residue sequence of β₃-endonexin is sufficient for specific binding to the β₃ cytoplasmic domain. These studies have identified a cytoplasmic domain sequence motif that specifies an integrin-specific protein-protein interaction.

Footnotes

↵^§ Supported by postdoctoral fellowships from the Deutsche Forschungsgemeinschaft and the American Heart Association (California affiliate). Present address: Medical Clinic, University of Würzburg, D-97080 Würzburg, Germany.
↵^¶ Supported by postdoctoral fellowships from the Austrian Fonds zur Förderung der wissenschaftlichen Forschung and the Swiss Nationalfonds.
↵* This work was supported by Grants HL 48728, AR 27214, and HL 56595 from the National Institutes of Health and by grants from Cor Therapeutics, Inc. This is publication 10305 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.
↵¹ The abbreviations used are:

CHO

Chinese hamster ovary

PAGE

polyacrylamide gel electrophoresis

PCR

polymerase chain reaction.
↵² H. Kashiwagi and S. J. Shattil, unpublished data.
- Received September 13, 1996.
- Revision received January 9, 1997.