Specific Association Of CD63 with the VLA-3 and VLA-6 Integrins

doi:10.1074/jbc.270.30.17784

Specific Association Of CD63 with the VLA-3 and VLA-6 Integrins (*)

From the Dana-Farber Cancer Institute, Harvard Medical School, Boston Massachusetts 02115

^§ To whom correspondence should be addressed: Dana-Farber Cancer Institute, Rm. M613, 44 Binney St., Boston, MA 02115. Tel.: 617-632-3410; Fax: 617-632-2662.

Abstract

We screened monoclonal antibodies to cell-surface proteins and selected an antibody, called 6H1, that recognizes a putative integrin-associated protein. The 6H1 monoclonal antibody (mAb) indirectly coprecipitated α³β₁ and/or α⁶β₁, but not α²β₁, or α⁵β₁ from Brij 96 detergent lysates of multiple cell lines. Large scale purification using the 6H1 mAb yielded a single protein of 45-60 kDa with an amino-terminal sequence that exactly matched CD63. Confirming that the 6H1 mAb recognized the CD63 protein, 6H1 and a known anti-CD63 mAb yielded identical coprecipitation results and identical colocalization into lysosomal granules containing cathepsin D. Furthermore, we used an established anti-CD63 mAb to detect this protein in an α³β₁ immunoprecipitate, and also we observed VLA-3 and CD63 colocalization in cellular “footprints.” Notably, the cytoplasmic domain of α³ was neither required nor sufficient for CD63 association, suggesting that it occurred elsewhere within the α³β₁ complex. Knowledge of these specific CD63-α³β₁ and CD63-α⁶β₁ biochemical associations should lead to critical insights into the specialized functions of α³β₁, α⁶β₁, and CD63.

Footnotes

↵* This work was supported by National Institutes of Health Grant GM46526, the Dana-Farber/Sandoz Drug Discovery Program, and a fellowship from the Sanofi Foundation for Thrombosis Research (Paris) (to G. B.). 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.
↵¹ The abbreviations used are:

mAb

monoclonal antibody

CHO

Chinese hamster ovary

FITC

fluorescein isothiocyanate

PBS

phosphate-buffered saline

DMEM

Dulbecco's modified Eagle's medium

PAGE

polyacrylamide gel electrophoresis

FBS

fetal bovine serum.
↵²G. Bazzoni and M. E. Hemler, submitted for publication.
↵³J. Weitzman, A. Chen, and M. E. Hemler, submitted for publication.
- Received March 20, 1995.
- Revision received April 26, 1995.