The Conserved Membrane-proximal Region of an Integrin Cytoplasmic Domain Specifies Ligand Binding Affinity (*)
- Paul E. Hughes(§),
- Timothy E. O'Toole(¶),
- Jari Ylänne(1),
- Sanford J. Shattil(2) and
- Mark H. Ginsberg(**)
- From the (1)Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037, the Department of Biochemistry, University of Helsinki, Helsinki 00170, Finland, and the
- (2)Department of Cell and Developmental Biology and Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
- ** To whom correspondence should be addressed. Tel.: 619-554-7124; Fax: 619-554-6403.
Abstract
Integrin affinities for ligands can change markedly via a process termed inside-out signaling. We expressed several truncations
of the β3 cytoplasmic domain in conjunction with an “activating” α subunit chimera, α
α
. Deletion of the 4 C-terminal residues of the β3 tail blocked inside-out signaling as assessed by the binding of an activation-specific antibody, PAC1. Several additional
truncations remained in the low affinity state, but complete truncation (β3Δ717) caused PAC1 binding. Activation by this truncation mutant did not depend on the α subunit cytoplasmic domain and was
resistant to inhibitors of cellular metabolism and the over-expression of an isolated β3 cytoplasmic domain. Since deletion of β3(Leu
-Asp
) results in a constitutively activated integrin, this membrane-proximal seven amino acids of the β3 cytoplasmic domain is required to maintain α
β3 in a default low affinity state. The amino acid sequence of this region is conserved among integrins. Moreover, the conserved
membrane-proximal sequence in α subunit tails seems to serve a similar function. Consequently, the conserved membrane-proximal
regions of both integrin cytoplasmic domains control the ligand binding affinity of the extracellular domain.
Footnotes
-
↵§ Post-doctoral fellow of the California Heart Association.
-
↵¶ Supported by TRDP 3RT0320 from the California Tobacco Diseases Research Program and an Established Investigator of the American Heart Association.
-
↵* 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. This work was supported by National Institutes of Health Grants HL48728 and AR 27214 (to M. H. G.) and HL 40387 (to S. J. S.). This is manuscript number 9116-VB from the Scripps Research Institute.
-
↵1 The abbreviations used are:
- CHO
-
Chinese hamster ovary
- LIBS
-
ligand-induced binding site
- DMEM
-
Dulbecco's modified Eagle's medium
- PCR
-
polyacrylamide chain reaction
- kb
-
kilobase(s).
-
- Received January 23, 1995.
- Revision received March 24, 1995.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
