Identifying the Putative Metal Ion-dependent Adhesion Site in the β2 (CD18) Subunit Required for αLβ2 and αMβ2 Ligand Interactions*
- ‡To whom correspondence should be addressed: Cell Biology and Inflammation Research, Pharmacia & Upjohn, 7239-209-219, 301 Henrietta St., Kalamazoo, MI 49001.
Abstract
We have previously demonstrated that Asp134 and Ser136 of the β2 subunit are essential for αLβ2 and αMβ2 ligand recognition. It has been proposed that these residues may be part of a metal ion-dependent adhesion site (MIDAS) within the β subunit homologous to the αM I domain MIDAS structure (Lee, J.-O., Rieu, P., Arnaout, M. A., and Liddington, R. (1995) Cell 80, 631-638). In the present study, we evaluated the role of additional candidate metal ion-coordinating residues in the β2 subunit in ligand interactions. Cells bearing the recombinant αLβ2 or αMβ2 mutant(s) were tested for the ability to bind to immobilized ligands. Alanine substitution at Asp232 in β2 produced a complete loss in the capacity of both αLβ2 and αMβ2 to support cell adhesion and suppressed the expression of a divalent cation-dependent conformation recognized by mAb 24. Alanine substitution at Glu235 differentially affected receptor function dependent upon the co-transfected α subunit. Cells expressing αLβ2 with a substitution at Glu235 failed to adhere to intercellular adhesion molecule 1 (ICAM-1) but did retain the capacity to bind mAb 24. Moreover, cells expressing αMβ2 with a substitution at Glu235 failed to adhere to fibrinogen or ICAM-1 and did not bind mAb 24. However, these cells did retain the capacity to adhere to iC3b following antibody-induced activation. These results implicate Asp232 and Glu235, along with Asp134 and Ser136, in ligand binding function of αLβ2 and αMβ2. These findings provide evidence in support of the existence of a MIDAS structure in β2 analogous to that seen in the αM I domain.
Footnotes
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↵* 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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↵1 The abbreviations used are:
- ICAM
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intercellular adhesion molecule
- MIDAS
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metal ion-dependent adhesion site
- mAb
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monoclonal antibody
- CHO
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Chinese hamster ovary
- TBS
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Tris-buffered saline
- FACS
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fluorescence-activated cell sorting.
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↵2R. P. Orchekowski and M. L. Bajt, submitted for publication.
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- Received February 12, 1996.
- Revision received July 18, 1996.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
