Site-directed mutagenesis of CCR2 identified amino acid residues in transmembrane helices 1, 2, and 7 important for MCP-1 binding and biological functions

    Authors

    M. A. Gavrilin; I. V. Gulina; T. Kawano; S. Dragan; L. Chakravarti;P. E. Kolattukudy

    Comments

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    Abbreviated Journal Title

    Biochem. Biophys. Res. Commun.

    Keywords

    monocytes/macrophages; chemokines; chemotaxis; signal transduction; MONOCYTE CHEMOTACTIC PROTEIN-1; CHEMOATTRACTANT PROTEIN-1; MOLECULAR-CLONING; ACTIVATING-FACTOR; LIGAND-BINDING; RECEPTOR; CHEMOKINES; PHOSPHORYLATION; INTERNALIZATION; DISSOCIATION; Biochemistry & Molecular Biology; Biophysics

    Abstract

    Monocyte chemotactic protein-1 (MCP-1) binds its G-protein-coupled seven transmembrane (TM) receptor. CCR2B, and causes infiltration of monocytes/macrophages into areas of injury, infection or inflammation. To identify functionally important amino acid residues in CCR2B, we made specific mutations of nine residues selected on the basis of conservation in chemokine receptors and located TM1 (Tyr(49)) TM2 (Leu(95)) TM3 (Thr(117) and Tyr(120)), and TM7 (Ala(286), Thr(290), Glu(291), and His(297)) and in the extracellular loop 3 (Glu(278)). MCP-1 binding was drastically affected only by mutations in TM7. Reversing the charge at Glu(291) (E291K) and a; His(297) (H297D) prevented MCP binding although substitution with Ala at either site had little effect, suggesting that Glu(291) and His(297) probably stabilize TM7 by their ionic interaction. E291A elicited normal Ca2+ influx. H297A. YA9F in TM1 and L95A in TM2 that showed normal MCP-1 binding did not elicit Ca2+ influx and elicited no adenylate cyclase inhibition at any MCP-1 concentration. MCP-1 treatment of HEK293 cells caused lamellipodia formation only when they expressed CCR2B. The mutants that showed no Ca2+ influx and adenylate cyclase inhibition by MCP-1 treatment showed lamellipodia formation and chemotaxis. Our results show that induction of lamellipodia formation. but not Ca2+ influx and adenylate cyclase inhibition, is necessary for chemotaxis. (C) 2004 Elsevier Inc. All rights reserved.

    Journal Title

    Biochemical and Biophysical Research Communications

    Volume

    327

    Issue/Number

    2

    Publication Date

    1-1-2005

    Document Type

    Article

    Language

    English

    First Page

    533

    Last Page

    540

    WOS Identifier

    WOS:000226364000021

    ISSN

    0006-291X

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