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Identification of CCR8: a human monocyte and thymus receptor for the CC chemokine I-309.

Tiffany HL, Lautens LL, Gao JL, Pease J, Locati M, Combadiere C, Modi W, Bonner TI, Murphy PM - J. Exp. Med. (1997)

Bottom Line: Signaling was sensitive to pertussis toxin, suggesting coupling to a Gi-type G protein.These properties parallel those of endogenous I-309 receptors expressed in an HL-60 clone 15 cell line model.The apparent monogamous relationship between I-309 and CCR8 is unusual among known CC chemokines and known CC chemokine receptors.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
The human CC chemokine I-309 is a potent monocyte chemoattractant and inhibits apoptosis in thymic cell lines. Here, we identify a specific human I-309 receptor, and name it CCR8 according to an accepted nomenclature system. The receptor has seven predicted transmembrane domains, is expressed constitutively in monocytes and thymus, and is encoded by a previously reported gene of previously unknown function named, alternatively, CY6, TER1, and CKR-L1. After transfection with the CY6 open reading frame, a mouse pre-B cell line exhibited calcium flux and chemotaxis in response to I-309 (EC50 = 2 nM for each), whereas 20 other chemokines were inactive. Signaling was sensitive to pertussis toxin, suggesting coupling to a Gi-type G protein. These properties parallel those of endogenous I-309 receptors expressed in an HL-60 clone 15 cell line model. The apparent monogamous relationship between I-309 and CCR8 is unusual among known CC chemokines and known CC chemokine receptors. CCR8 may regulate monocyte chemotaxis and thymic cell line apoptosis.

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I-309 is an agonist  for CCR8. (A) Receptor specificity and homologous densensitization. [Ca2+]i was monitored by  ratio fluorescence of Fura-2–loaded  pre–B cells or HEK 293 cells stably transfected with plasmids encoding CC chemokine receptors  as indicated adjacent to each  tracing. Cells were stimulated  with chemokines 50 nM at the  times indicated by arrowheads.  Data are representative of at least  three experiments with CCR8-expressing cells. (B) Potency.  The amplitude of the peak of the  calcium transient elicited by the  indicated concentration of I-309  in CCR8 transfectants is shown.  Data are representative of two  separate experiments.
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Figure 2: I-309 is an agonist for CCR8. (A) Receptor specificity and homologous densensitization. [Ca2+]i was monitored by ratio fluorescence of Fura-2–loaded pre–B cells or HEK 293 cells stably transfected with plasmids encoding CC chemokine receptors as indicated adjacent to each tracing. Cells were stimulated with chemokines 50 nM at the times indicated by arrowheads. Data are representative of at least three experiments with CCR8-expressing cells. (B) Potency. The amplitude of the peak of the calcium transient elicited by the indicated concentration of I-309 in CCR8 transfectants is shown. Data are representative of two separate experiments.

Mentions: To identify a specific agonist, we screened a panel of chemoattractants for the ability to induce calcium flux in the mouse pre-B cell line 4DE4 before and after transfection with a plasmid encoding CCR8. Untransfected 4DE4 cells did not respond to any agonists tested except for the CXC chemokine SDF-1 (Fig. 2 A). 4DE4 cells transfected with the CCR8 plasmid exhibited [Ca2+]i transients in response to SDF-1 and I-309, but not in response to the following tested at 50 nM or greater: the CC chemokines HCC-1, MIP-1α, RANTES, MIP-1β, MCP-1, MCP-2, MCP-3, MCP-4, and eotaxin; the CXC chemokines IL-8, γIP-10, NAP-2, GRO-α, GRO-β, GRO-γ, and ENA-78; the C chemokine lymphotactin; and the nonchemokine leukocyte chemoattractants fMLP, C3a, and C5a (data not shown). I-309 did not induce calcium flux in 4DE4 cell lines expressing CCR1 or CCR3, or in HEK 293 cells stably expressing CCR5 (Fig. 2 A; data not shown). The CCR1, CCR3, and CCR5 cell lines all responded appropriately to previously described agonists (Fig. 2 A; data not shown).


Identification of CCR8: a human monocyte and thymus receptor for the CC chemokine I-309.

Tiffany HL, Lautens LL, Gao JL, Pease J, Locati M, Combadiere C, Modi W, Bonner TI, Murphy PM - J. Exp. Med. (1997)

I-309 is an agonist  for CCR8. (A) Receptor specificity and homologous densensitization. [Ca2+]i was monitored by  ratio fluorescence of Fura-2–loaded  pre–B cells or HEK 293 cells stably transfected with plasmids encoding CC chemokine receptors  as indicated adjacent to each  tracing. Cells were stimulated  with chemokines 50 nM at the  times indicated by arrowheads.  Data are representative of at least  three experiments with CCR8-expressing cells. (B) Potency.  The amplitude of the peak of the  calcium transient elicited by the  indicated concentration of I-309  in CCR8 transfectants is shown.  Data are representative of two  separate experiments.
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Figure 2: I-309 is an agonist for CCR8. (A) Receptor specificity and homologous densensitization. [Ca2+]i was monitored by ratio fluorescence of Fura-2–loaded pre–B cells or HEK 293 cells stably transfected with plasmids encoding CC chemokine receptors as indicated adjacent to each tracing. Cells were stimulated with chemokines 50 nM at the times indicated by arrowheads. Data are representative of at least three experiments with CCR8-expressing cells. (B) Potency. The amplitude of the peak of the calcium transient elicited by the indicated concentration of I-309 in CCR8 transfectants is shown. Data are representative of two separate experiments.
Mentions: To identify a specific agonist, we screened a panel of chemoattractants for the ability to induce calcium flux in the mouse pre-B cell line 4DE4 before and after transfection with a plasmid encoding CCR8. Untransfected 4DE4 cells did not respond to any agonists tested except for the CXC chemokine SDF-1 (Fig. 2 A). 4DE4 cells transfected with the CCR8 plasmid exhibited [Ca2+]i transients in response to SDF-1 and I-309, but not in response to the following tested at 50 nM or greater: the CC chemokines HCC-1, MIP-1α, RANTES, MIP-1β, MCP-1, MCP-2, MCP-3, MCP-4, and eotaxin; the CXC chemokines IL-8, γIP-10, NAP-2, GRO-α, GRO-β, GRO-γ, and ENA-78; the C chemokine lymphotactin; and the nonchemokine leukocyte chemoattractants fMLP, C3a, and C5a (data not shown). I-309 did not induce calcium flux in 4DE4 cell lines expressing CCR1 or CCR3, or in HEK 293 cells stably expressing CCR5 (Fig. 2 A; data not shown). The CCR1, CCR3, and CCR5 cell lines all responded appropriately to previously described agonists (Fig. 2 A; data not shown).

Bottom Line: Signaling was sensitive to pertussis toxin, suggesting coupling to a Gi-type G protein.These properties parallel those of endogenous I-309 receptors expressed in an HL-60 clone 15 cell line model.The apparent monogamous relationship between I-309 and CCR8 is unusual among known CC chemokines and known CC chemokine receptors.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
The human CC chemokine I-309 is a potent monocyte chemoattractant and inhibits apoptosis in thymic cell lines. Here, we identify a specific human I-309 receptor, and name it CCR8 according to an accepted nomenclature system. The receptor has seven predicted transmembrane domains, is expressed constitutively in monocytes and thymus, and is encoded by a previously reported gene of previously unknown function named, alternatively, CY6, TER1, and CKR-L1. After transfection with the CY6 open reading frame, a mouse pre-B cell line exhibited calcium flux and chemotaxis in response to I-309 (EC50 = 2 nM for each), whereas 20 other chemokines were inactive. Signaling was sensitive to pertussis toxin, suggesting coupling to a Gi-type G protein. These properties parallel those of endogenous I-309 receptors expressed in an HL-60 clone 15 cell line model. The apparent monogamous relationship between I-309 and CCR8 is unusual among known CC chemokines and known CC chemokine receptors. CCR8 may regulate monocyte chemotaxis and thymic cell line apoptosis.

Show MeSH
Related in: MedlinePlus