Probing the function of LFA-1 using fluorescent proteins that target the beta-2 integrin transmembrane domain

The lymphocyte functional antigen-1 (LFA-1) is a type I heterodimeric transmembrane (TM) proteins involved in cell adhesion, and mediates a number of cellular and physiological processes. In this work, we used recombinant fluorescently-tagged proteins derived from the TM domain of the β ₂ integrin to disrupt the function of LFA-1 on Jurkat cells. Four variants of the proteins were made including: one with a short cytoplasmic tail (EGFPβ ₂TM+CD), without the cytoplasmic tail (EGFPβ₂TM-CD), truncation of five amino acids (EGFPβ₂TM- 5B) and truncation of ten amino acid (EGFPβ₂TM-10B). These proteins were able to label Jurkat cells in vitro in a protein binding assay with affinity, K_d as high as 280 ± 80 nM (EGFPβ ₂TM-5B). We used fluosphere beads conjugated to different mAb to study the effect of binding on the epitopes of LFA-1. The proteins had an overall activation effect on MEM148 and an inhibitory effect on MEM48 epitope of LFA-1 receptor