Interaction of calmodulin with L-selectin at the membrane interface: implication on the regulation of L-selectin shedding |
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Authors: | Deng Wei Srinivasan Sankaranarayanan Zheng Xiaofeng Putkey John A Li Renhao |
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Affiliation: | Center for Membrane Biology, Department of Biochemistry and Molecular Biology, The University of Texas Health Science Center at Houston, Houston, TX 77030, USA |
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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. |
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Keywords: | CaM, calmodulin CaMKII, CaM-dependent protein kinase II D/A-CaM, donor/acceptor-labeled CaM DDPM, N-(4-dimethylamino-3,5-dinitrophenyl)maleimide EGFR, epidermal growth factor receptor HSQC, heteronuclear single quantum coherence IAEDANS, 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid ITC, isothermal titration calorimetry POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine POPS, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho- smallcaps" >l-serine PS, phosphatidylserine TMR, tetramethylrhodamine-5-maleimide EDTA, ethylenediaminetetraacetic acid |
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