The Cytoplasmic Domain of L-Selectin Participates in Regulating L-Selectin Endoproteolysis

• Published in: The Journal of immunology (1950) Vol. 167; no. 3; pp. 1617 - 1623
• Main Authors: Matala, Erik, Alexander, Shelia R, Kishimoto, Takashi K, Walcheck, Bruce
• Format: Journal Article
• Language: English
• Published: United States Am Assoc Immnol 01.08.2001
• Subjects: Amino Acid Sequence; Calmodulin - metabolism; Cell Line; Cell Membrane - metabolism; Cytoplasm - metabolism; Cytoplasm - physiology; Endopeptidases - metabolism; Humans; Hydrolysis; Interphase - physiology; K562 Cells; L-selectin; L-Selectin - genetics; L-Selectin - metabolism; L-Selectin - physiology; Molecular Sequence Data; Mutagenesis, Site-Directed; Neutrophils - metabolism; Peptide Fragments - genetics; Peptide Fragments - metabolism; Peptide Fragments - physiology; Protein Binding - genetics; Protein Structure, Tertiary - genetics; Protein Structure, Tertiary - physiology; Transfection
• ISSN: 0022-1767; 1550-6606
• DOI: 10.4049/jimmunol.167.3.1617

Neutrophil recruitment at sites of inflammation is regulated by a series of adhesion and activation events. L-selectin (CD62L) is a leukocyte expressed adhesion protein that is important for neutrophil accumulation and rolling along the vascular endothelium. L-selectin is unique from other adhesion molecules involved in leukocyte transmigration in that its adhesiveness appears to be regulated partly by rapid endoproteolysis. Cleavage of L-selectin occurs within a membrane-proximal region that results in ectodomain shedding and retention of a 6-kDa transmembrane fragment. The cleavage domain of L-selectin has been well characterized through mutational analysis. Whether the cytoplasmic domain of L-selectin also plays a role in regulating shedding is controversial. We have previously shown that the Ca(2+)-sensing protein calmodulin (CaM) constitutively associates with the cytoplasmic domain of L-selectin in transfected cell lines. However, in the absence of mapping and mutational analysis of the CaM-binding region of L-selectin, there remains no direct evidence that this interaction affects shedding. Using synthesized 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 increase the proteolytic turnover of L-selectin. Moreover, we demonstrate that CaM binding to the 6-kDa transmembrane fragment is greatly reduced compared with intact L-selectin in neutrophils, suggesting that CaM binding is regulated. These data imply that the cytoplasmic domain of L-selectin can regulate shedding by a mechanism in which bound CaM may operate as a negative effector.