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

Keywords

Chemokines; Chemotaxis; Monocytes/macrophages; Signal transduction

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 at 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 Ca 2+ influx. H297A, Y49F in TM1 and L95A in TM2 that showed normal MCP-1 binding did not elicit Ca 2+ 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 Ca 2+ influx and adenylate cyclase inhibition by MCP-1 treatment showed lamellipodia formation and chemotaxis. Our results show that induction of lamellipodia formation, but not Ca 2+ influx and adenylate cyclase inhibition, is necessary for chemotaxis. © 2004 Elsevier Inc. All rights reserved.

Publication Date

2-11-2005

Publication Title

Biochemical and Biophysical Research Communications

Volume

327

Issue

2

Number of Pages

533-540

Document Type

Article

Personal Identifier

scopus

DOI Link

https://doi.org/10.1016/j.bbrc.2004.12.037

Socpus ID

11144292658 (Scopus)

Source API URL

https://api.elsevier.com/content/abstract/scopus_id/11144292658

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