Using photo‐crosslinking to determine intracellular CD11b/CD18 signaling interactions (609.12)

Abstract

The leukocyte integrin, Mac‐1 (CD11b/CD18), plays an essential role in mediating both cell‐cell and cell‐extracellular matrix adhesion interactions during an inflammatory response. Historically, integrin signaling was understood to be both bidirectional, through inside‐out and outside‐in signaling, and involve either activation or deactivation of the integrin. However, more recent studies suggest a non‐binary activation pathway for Mac‐1, which results in a complex and differential signal transduction pattern. In order to understand the implications of a non‐binary activation of CD11b/CD18, direct protein interactions must be identified and quantified; however, determining direct interactions continues to be a challenge in the field of proteomics. Herein, we report our use of the unnatural amino acid (UAA), benzophenone, incorporated at several relevant sites in the cytoplasmic tails of Mac‐1 to photocrosslink proteins that directly interact with Mac‐1 under various physiological conditions. Plasmids coding for amber stop codons at the desired amino acid sites were prepared using PCR mediated site‐directed mutagenesis. HEK293 and CHO cell lines were transiently transfected and grown under appropriate physiological conditions in the presence of the unnatural amino acid, benzophenone. The cells were irradiated with UV light and the resulting photo‐crosslinked proteins were identified using western blot analysis. Our results demonstrate that photocrosslinking using UAA is a powerful tool for identifying protein interactions involved in signal transduction. Further analysis will enable us to more thoroughly understand the complex signaling process involved with the Mac‐1 integrin during leukocytic inflammation.