title: Interaction of calmodulin with L-selectin at the membrane interface: implication on the regulation of L-selectin shedding.
abstract: The calmodulin (CaM) hypothesis of ectodomain shedding stipulates that CaM, an intracellular Ca²⁺-dependent regulatory protein, associates with the cytoplasmic domain of L-selectin to regulate ectodomain shedding of L-selectin on the other side of the plasma membrane. To understand the underlying molecular mechanism, we have characterized the interactions of CaM with two peptides derived from human L-selectin. The peptide ARR18 corresponds to the entire cytoplasmic domain of L-selectin (residues Ala317-Tyr334 in the mature protein), and CLS corresponds to residues Lys280-Tyr334, which contains the entire transmembrane and cytoplasmic domains of l-selectin. Monitoring the interaction by fluorescence spectroscopy and other biophysical techniques, we found that CaM can bind to ARR18 in aqueous solutions or the L-selectin cytoplasmic domain of CLS reconstituted in the phosphatidylcholine bilayer, both with an affinity of approximately 2 μM. The association is calcium independent and dynamic and involves both lobes of CaM. In a phospholipid bilayer, the positively charged L-selectin cytoplasmic domain of CLS is associated with anionic phosphatidylserine (PS) lipids at the membrane interface through electrostatic interactions. Under conditions where the PS content mimics that in the inner leaflet of the cell plasma membrane, the interaction between CaM and CLS becomes undetectable. These results suggest that the association of CaM with L-selectin in the cell can be influenced by the membrane bilayer and that anionic lipids may modulate ectodomain shedding of transmembrane receptors.
authors.affiliation: Center for Membrane Biology, Department of Biochemistry and Molecular Biology, The University of Texas Health Science Center at Houston, Houston, TX 77030, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Deng
authors.forename: Sankaranarayanan
authors.initials: S
authors.lastname: Srinivasan
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Zheng
authors.forename: John A
authors.initials: JA
authors.lastname: Putkey
authors.forename: Renhao
authors.initials: R
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D002147:Calmodulin / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D019041:L-Selectin / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D008969:Molecular Sequence Data; D010718:Phosphatidylserines / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping; D013050:Spectrometry, Fluorescence; D055672:Static Electricity
chemical_list: D002147:Calmodulin; D008081:Liposomes; D010718:Phosphatidylserines; D019041:L-Selectin