MEMBRANE PROXIMAL CLEAVAGE OF L-SELECTIN - IDENTIFICATION OF THE CLEAVAGE SITE AND A 6-KD TRANSMEMBRANE PEPTIDE FRAGMENT OF L-SELECTIN

Rapid downregulation of L-selectin expression occurs in response to le ukocyte activation, and it has been speculated to be an integral proce ss in the adhesion cascade leading to neutrophil recruitment to sites of inflammation. It has previously been proposed that L-selectin is pr oteolytically cleaved from the cell surface; however, the nature of th e cleavage site has been unknown. We have produced polyclonal antisera against the extracellular domain and against the cytoplasmic domain o f L-selectin. Both antisera immunoprecipitate the intact form of L-sel ectin from metabolically labeled phytohemagglutinin-stimulated lymphob lasts and peripheral blood neutrophils. In addition, the anti-cytoplas mic domain serum, but not the antiectodomain serum, immunoprecipitate a 6-kD species from PMA activated lymphoblasts and formyl-methionylleu cylphenylalanine-activated neutrophils. Conversely, the antiectodomain serum but not the anti-cytoplasmic domain serum immunoprecipitate a 6 8-kD soluble form of L-selectin from the supernatant of PMA-activated lymphoblasts. The appearance of the 6-kD species on activated cells co rrelated with the disappearance of the intact form of L-selectin and t he appearance of the soluble form of L-selectin. A third polyclonal se rum generated against the membrane proximal region of the ectodomain a lso reacted with the 6-kD species, indicating that this is a transmemb rane peptide of L-selectin. That the 6-kD species is derived from L-se lectin was confirmed by immunoprecipitation of the 6-kD species from L -selectin transfectants but not from mock transfectants. Radiochemical sequence analysis defined a cleavage site between Lys321 and Ser322, which would predict a transmembrane fragment consistent in size with t he observed 6-kD fragment. A Ser-Phe-Ser motif adjacent to the cleavag e site is conserved between human, mouse, and rat L-selectin, and a re lated motif is found proximal to transmembrane domains of other downre gulated proteins, such as ACE, CD16-II, and TNF-RII, suggesting the po ssibility of a common recognition motif.