Osteopontin N-terminal Domain Contains a Cryptic Adhesive Sequence Recognized by α9β1 Integrin

doi:10.1074/jbc.271.45.28485

Osteopontin N-terminal Domain Contains a Cryptic Adhesive Sequence Recognized by α₉β₁ Integrin*

From the Department of Pathology, University of Washington, Seattle, Washington 98195,
^§ Biogen, Department of Biological Research, Cambridge, Massachusetts 02142,
the ^¶ Department of Medicine, University of California, San Francisco, California 94143-0854, and
^¶ Lung Biology Center, San Francisco General Hospital, San Francisco, California 94110-3594

** An established investigator of the American Heart Association. To whom correspondence and reprint requests should be addressed:
Pathology Dept., University of Washington, Vascular Biology, Box 357335, Seattle, WA 98195
. Tel. 206-543-0205; Fax: 206-685-3662; E-mail: ceci{at}u.washington.edu.

Abstract

Osteopontin is an adhesive glycoprotein implicated in numerous diseases associated with inflammation and remodeling. There are several structural domains in osteopontin that are of particular interest. The RGD motif is a cell attachment sequence shown to be critical for cell adhesion through α_v-containing integrins. In close proximity to the RGD domain is the thrombin cleavage site. Previous observations suggest that thrombin cleavage of osteopontin occurs in vivo and may be physiologically important. To study the functional significance of osteopontin cleavage by thrombin, we made glutathione S-transferase-osteopontin fusion proteins. These proteins contain either the N- or C-terminal domains expected to be formed following thrombin cleavage at the Arg¹⁶⁹-Ser¹⁷⁰ peptide bond. We compared these osteopontin fragments with native osteopontin in their ability to support adhesion of several different cell lines and identified the receptors mediating these interactions. Our data show that the N-terminal osteopontin fragment, which contains the RGD domain, supports adhesion of a melanoma cell line that is unable to bind native osteopontin. This suggests that osteopontin adhesive interactions may be regulated by thrombin cleavage. We also demonstrate that osteopontin contains a cryptic binding activity, which can be recognized by a novel osteopontin receptor. This receptor has been identified as the α₉β₁ integrin.

Footnotes

↵^‡ Supported by NIH Grant 5T32 GM07270-20.
↵* This study was supported by National Institutes of Health (NIH) Grant HL-18645. 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:

GST

glutathione S-transferase

OPN

osteopontin

mAb

monoclonal antibody

PAGE

polyacrylamide gel electrophoresis

BSA

bovine serum albumin

PBS

phosphate-buffered saline

FACS

fluorescence-activated cell sorting.
↵² L. L. Smith, H.-K. Cheung, L. E. Ling, J. Chen, D. Sheppard, R. Pytela, and C. M. Giachelli, unpublished observations.
- Received May 30, 1996.
- Revision received August 15, 1996.