Constitutive activation of CCR5 and CCR2 induced by conformational changes in the conserved TXP motif in transmembrane helix 2

Diana Alvarez Arias, Jean Marc Navenot, Wenbo Zhang, James Broach, Stephen C. Peiper

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

CCR5 is a G protein-coupled receptor for RANTES, MIP-α, MIP-1β, and MCP-2 that functions as the front line coreceptor for human immunodeficiency virus type 1 infection. To elucidate the mechanism for CCR5 activation, this coreceptor was expressed in yeast coupled to the pheromone response pathway and a constitutively active mutant (CAM) was derived by random mutagenesis. Conversion of Thr-82 in the highly conserved TXP motif in transmembrane helix 2 to Pro, His, Tyr, Arg, or Lys conferred autonomous signaling activity in yeast and mammalian cells. This substitution also imparted constitutive signaling to CCR2 in yeast and mammalian cells, but not CCR1, CCR3, CCR4, CXCR2, or CXCR4. The CCR5-CAM, but not the CCR2-CAM had a reduction in ligand binding affinity. Whereas the amplitude of calcium mobilization induced by RANTES stimulation was lower in the CCR5-CAM than the wild-type (WT) receptor, MCP-1 induced a higher signal in the CCR2-CAM than in CCR2-WT. The chemotactic response of CCR5-CAM(T82P) to RANTES was similar to that of CCR5-WT, but CCR5-CAM(T82K) was dramatically decreased. The chemotactic response of CCR2-WT and CCR2-CAM(T94K) were similar. These findings extend insight into the role of the TXP motif in the mechanism for CCR5 signaling. CCR2, the receptor most closely genetically related to CCR5, shared a similar signaling mechanism, but other receptors containing the TXP motif did not. The expression of CCR5 and CCR2 in yeast and the availability of variants with autonomous signaling represent critical tools for characterizing receptor antagonists and developing approaches to block their role in human diseases.

Original languageEnglish (US)
Pages (from-to)36513-36521
Number of pages9
JournalJournal of Biological Chemistry
Volume278
Issue number38
DOIs
StatePublished - Sep 19 2003
Externally publishedYes

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Chemokine CCL5
Yeasts
CCR2 Receptors
Chemical activation
Yeast
Cells
Pheromones
Virus Diseases
G-Protein-Coupled Receptors
Mutagenesis
HIV-1
Viruses
Ligands
Calcium
Substitution reactions
Availability

ASJC Scopus subject areas

  • Biochemistry

Cite this

Constitutive activation of CCR5 and CCR2 induced by conformational changes in the conserved TXP motif in transmembrane helix 2. / Arias, Diana Alvarez; Navenot, Jean Marc; Zhang, Wenbo; Broach, James; Peiper, Stephen C.

In: Journal of Biological Chemistry, Vol. 278, No. 38, 19.09.2003, p. 36513-36521.

Research output: Contribution to journalArticle

Arias, Diana Alvarez ; Navenot, Jean Marc ; Zhang, Wenbo ; Broach, James ; Peiper, Stephen C. / Constitutive activation of CCR5 and CCR2 induced by conformational changes in the conserved TXP motif in transmembrane helix 2. In: Journal of Biological Chemistry. 2003 ; Vol. 278, No. 38. pp. 36513-36521.
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abstract = "CCR5 is a G protein-coupled receptor for RANTES, MIP-α, MIP-1β, and MCP-2 that functions as the front line coreceptor for human immunodeficiency virus type 1 infection. To elucidate the mechanism for CCR5 activation, this coreceptor was expressed in yeast coupled to the pheromone response pathway and a constitutively active mutant (CAM) was derived by random mutagenesis. Conversion of Thr-82 in the highly conserved TXP motif in transmembrane helix 2 to Pro, His, Tyr, Arg, or Lys conferred autonomous signaling activity in yeast and mammalian cells. This substitution also imparted constitutive signaling to CCR2 in yeast and mammalian cells, but not CCR1, CCR3, CCR4, CXCR2, or CXCR4. The CCR5-CAM, but not the CCR2-CAM had a reduction in ligand binding affinity. Whereas the amplitude of calcium mobilization induced by RANTES stimulation was lower in the CCR5-CAM than the wild-type (WT) receptor, MCP-1 induced a higher signal in the CCR2-CAM than in CCR2-WT. The chemotactic response of CCR5-CAM(T82P) to RANTES was similar to that of CCR5-WT, but CCR5-CAM(T82K) was dramatically decreased. The chemotactic response of CCR2-WT and CCR2-CAM(T94K) were similar. These findings extend insight into the role of the TXP motif in the mechanism for CCR5 signaling. CCR2, the receptor most closely genetically related to CCR5, shared a similar signaling mechanism, but other receptors containing the TXP motif did not. The expression of CCR5 and CCR2 in yeast and the availability of variants with autonomous signaling represent critical tools for characterizing receptor antagonists and developing approaches to block their role in human diseases.",
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