Mutational Analysis of Cell Cycle Inhibition by Integrin β1C*
Abstract
Integrin β1C is an alternatively spliced cytoplasmic variant of the β1 subunit that potently inhibits cell cycle progression. In this study, we analyzed the requirements for growth suppression by β1C. A chimera containing the extracellular/transmembrane domain of the Tac subunit of the human interleukin 2 receptor (gp55) fused to the cytoplasmic domain of β1C (residues 732–805) strongly inhibited growth in mouse 10T1/2 cells even at low expression levels, whereas chimeras containing the β1A, β1B, β1D, β3, and β5 cytoplasmic domains had weak and variable effects. The β1C cytoplasmic domain is composed of a membrane proximal region (732–757) common to all β1variants and a COOH-terminal 48-amino acid domain (758–805) unique to β1C. The β1C-specific domain (758–805) was sufficient to block cell growth even when expressed as a soluble cytoplasmic green fluorescent protein fusion protein. These results indicate that growth inhibition by β1C does not require the intact receptor and can function in the absence of membrane targeting. Analysis of deletions within the β1C-specific domain showed that the 18-amino acid sequence 775–792 is both necessary and sufficient for maximal growth inhibition, although the 13 COOH-terminal residues (793–805) also had weak activity. Finally, β1C is known to be induced in endothelial cells in response to tumor necrosis factor and is down-regulated in prostate epithelial cells after transformation. The green fluorescent protein/β1C (758–805) chimera blocked growth in the human endothelial cell line EV304 and in the transformed prostate epithelial cell line DU145, consistent with a role for β1C as a growth inhibitor in vivo.
Footnotes
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↵* This work was supported by National Institutes of Health Grants P01 HL48728 (to M. A. S.), R01 GM47214 (to M. A. S.), and GM47157 (to Y. T.) and by National Institutes of Health Training Grant T32 HL07695.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.
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↵‡ A research fellow of the American Heart Association, California Affiliate.
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↵§ To whom correspondence should be addressed: Dept. of Vascular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-784-7137; Fax: 619-784-7360; E-mail:schwartz{at}scripps.edu.
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↵2 D. Calderwood, C. Fenczik, and M. H. Ginsberg, manuscript in preparation.
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↵3 J. Meredith and M. A. Schwartz, unpublished observations.
- Abbreviations:
- aa
-
amino acid(s)
- IL2
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interleukin 2
- IL2R
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IL2 receptor
- GFP
-
green fluorescent protein
- BrdUrd
-
bromodeoxyuridine
- PBS
-
phosphate-buffered saline
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- Received November 11, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
