During an inflammatory response, leukocytes utilize a sequential series of steps to migrate into underlying tissues. The first step of this complex process enables leukocytes to tether and roll along the lumen of the blood vessel, an interaction mediated by endothelial selectins binding to their leukocyte ligands. Leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) plays a crucial role in this process. Many of the structural modifications of the PSGL-1 extracellular domain required for interactions with endothelial P-selectin are known. The importance of the highly conserved PSGL-1 cytoplasmic domain in the process of leukocyte rolling and migration is poorly understood. Our research has shown that interactions between the actin cytoskeleton and the PSGL-1 cytoplasmic domain are required for leukocyte rolling. We have also shown that ERM (ezrin/radixin/moesin) proteins are involved in these interactions. To further characterize the structural and functional importance of these interactions, we generated stable transfectants expressing PSGL-1 cytoplasmic domain truncations and point mutations. Analysis of these transfectants indicated that the PSGL-1 membrane proximal 17 amino acids are critical for interactions with ERM proteins. Conservative substitutions of positively charged residues within this 17 amino acid sequence inhibited these interactions whereas replacement of tyrosine residues with phenylalanine had no effect. Functional analysis of the stable transfectants will determine if interactions with ERM proteins are required for rolling on P-selectin. (This research is supported by NIH R01 GM60563 and T32HL007829)