Title

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

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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