The cytoplasmic domain of L-selectin participates in regulating L-selectinendoproteolysis

Neutrophil recruitment at sites of inflammation is regulated by a series of adhesion and activation events. L-selectin (CD62L) is a leukocyte expresse d adhesion protein that is important for neutrophil accumulation and rollin g along the vascular endothelium. L-selectin is unique from other adhesion molecules involved in leukocyte transmigration in that its adhesiveness app ears to be regulated partly by rapid endoproteolysis. Cleavage of L-selecti n occurs within a membrane-proximal region that results in ectodomain shedd ing and retention of a 6-kDa transmembrane fragment. The cleavage domain of L-selectin has been well characterized through mutational analysis. Whethe r the cytoplasmic domain of L-selectin also plays a role in regulating shed ding is controversial. We have previously shown that the Ca2+-sensing prote in calmodulin (CaM) constitutively associates with the cytoplasmic domain o f L-selectin in transfected cell lines. However, in the absence of mapping and mutational analysis of the CaM-binding region of L-selectin, there rema ins no direct evidence that this interaction affects shedding. Using synthe sized peptides and expressed L-selectin constructs, we demonstrate that CaM binding activity occurs in the membrane-proximal region of the cytoplasmic domain. Mutations engineered in this region that prevent CaM binding incre ase the proteolytic turnover of L-selectin. Moreover, we demonstrate that C aM binding to the 6-kDa transmembrane fragment is greatly reduced compared with intact L-selectin in neutrophils, suggesting that CaM binding is regul ated. These data imply that the cytoplasmic domain of L-selectin can regula te shedding by a mechanism in which bound CaM may operate as a negative eff ector.