Identification of CCR6, the Specific Receptor for a Novel Lymphocyte-directed CC Chemokine LARC

Masataka Baba,Toshio Imai,Miyuki Nishimura,Mayumi Kakizaki,Shin Takagi,Kunio Hieshima,Hisayuki Nomiyama,Osamu Yoshie

doi:10.1074/jbc.272.23.14893

Masataka Baba, Toshio Imai + Show 6 more

Open Access

https://doi.org/10.1074/jbc.272.23.14893

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Abstract

Liver and activation-regulated chemokine (LARC) is a recently identified CC chemokine that is expressed mainly in the liver. LARC functions as a selective chemoattractant for lymphocytes that express a class of receptors specifically binding to LARC with high affinity. To identifiy the receptor for LARC, we examined LARC-induced calcium mobilization in cells stably expressing five CC chemokine receptors (CCR1-CCR5) and five orphan seven-transmembrane receptors. LARC specifically induced calcium flux in K562 cells as well as 293/EBNA-1 cells stably expressing an orphan receptor GPR-CY4. LARC induced migration in 293/EBNA-1 cells stably expressing GPR-CY4 with a bi-modal dose-response curve. LARC fused with secreted alkaline phosphatase (LARC-SEAP) bound specifically to Raji cells stably expressing GPR-CY4 with a Kd of 0.9 nM. Only LARC but not five other CC chemokines (MCP-1, RANTES, MIP-1alpha, MIP-1beta, and TARC) competed with LARC-SEAP for binding to GPR-CY4. By Northern blot analysis, GPR-CY4 mRNA was expressed mainly in spleen, lymph nodes, Appendix, and fetal liver among various human tissues. Among various leukocyte subsets, GPR-CY4 mRNA was detected in lymphocytes (CD4(+) and CD8(+) T cells and B cells) but not in natural killer cells, monocytes, or granulocytes. Expression of GPR-CY4 mRNA in CD4(+) and CD8(+) T cells was strongly up-regulated by IL-2. Taken together, GPR-CY4 is the specific receptor for LARC expressed selectively on lymphocytes, and LARC is a unique functional ligand for GPR-CY4. We propose GPR-CY4 to be designated as CCR6.

Highlights

The chemokines are a group of structurally related approximately 70 –90-amino acid polypeptides involved in leukocyte recruitment and activation [1, 2]
Binding of Liver and activation-regulated chemokine (LARC)—Previously, we showed that LARC-SEAP(His)6 bound to a single class of receptors expressed on lymphocytes with a Kd of 0.4 nM [28]
The present study has further demonstrated that LARC is constitutively expressed at relatively low levels in tissues such as the lymph nodes, appendix, and fetal liver (Fig. 4B)

Summary

Introduction

The chemokines are a group of structurally related approximately 70 –90-amino acid polypeptides involved in leukocyte recruitment and activation [1, 2]. Induction of Calcium Mobilization by LARC—To examine interaction of LARC with each cloned receptor, we measured LARC-induced calcium mobilization in a panel of K562 cells stably expressing the five known CCRs (CCR1-CCR5) and five orphan chemokine receptors, V28/CMKBRL1 [31, 32], EBI1 [33], BLR1 [34], GPR-CY42 These chemokines, properly induced calcium flux in K562 cells expressing their respective CCRs even after treatment with LARC (Fig. 1A).

Results

Conclusion