The cytoplasmic tail of L-selectin interacts with members of the Ezrin-Radixin-Moesin (ERM) family of proteins: cell activation-dependent binding of Moesin but not Ezrin.
L-selectin regulates the recruitment of naive lymphocytes from the bloodstream to secondary lymphoid organs, mediating their initial capture and subsequent rolling along high endothelial cell surface-expressed ligands in peripheral lymph nodes. In vivo, distribution of L-selectin and cell surface levels determine the tethering efficiency and rolling velocity of leukocytes, respectively. Treatment of naive lymphocytes with phorbol myristate acetate (PMA) induces rapid ectodomain proteolytic down-regulation (shedding) of surface L-selectin via a protein kinase C (PKC)-dependent pathway. In an attempt to isolate proteins that are involved in regulating L-selectin expression, an affinity column was constructed using the 17-amino acid cytoplasmic tail of L-selectin. Affinity purification of extracts from lymphocytes, pre-treated with or without PMA, allowed identification of proteins that interact with the affinity column under one condition but not the other. By using this approach, members of the Ezrin-Radixin-Moesin family of proteins were found to interact specifically with the cytoplasmic tail of L-selectin. Moesin from PMA-stimulated lymphocytes, but not from unstimulated lymphocytes, bound to L-selectin tail. In contrast, ezrin from unstimulated or PMA-stimulated lymphocytes associated with L-selectin tail with equal affinity. Furthermore, the PKC inhibitor Ro 31-8220 significantly reduced the interaction of moesin, but not ezrin, with L-selectin. Alanine mutations of membrane-proximal basic amino acid residues in the cytoplasmic domain of L-selectin identified arginine 357 as a critical residue for both ezrin and moesin interaction. Finally, BIAcore affinity analysis confirmed that N-terminal moesin interacts specifically with L-selectin cytoplasmic tail, with relatively high affinity (K(d) approximately 40 nm). Based on these findings, although moesin and ezrin bind to a similar region of the cytoplasmic tail of L-selectin, moesin binding is dependent on PKC activation, which suggests that ezrin and moesin are regulated differently in lymphocytes.
Ivetic
Aleksandar
A
Divisions of Cellular Immunology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom.
Deka
Jürgen
J
Ridley
Anne
A
Ager
Ann
A
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D003593:Cytoplasm / Q000378:metabolism*; D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D019041:L-Selectin / Q000737:chemistry / Q000378:metabolism*; D008214:Lymphocytes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008840:Microfilament Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D025581:Neurofibromin 2 / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D000099149:Ezrin
D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors; D007211:Indoles; D019041:L-Selectin; D008840:Microfilament Proteins; D025581:Neurofibromin 2; D010750:Phosphoproteins; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D000099149:Ezrin; C071058:moesin; C064758:Ro 31-8220