Expression and purification of human FROUNT, a common cytosolic regulator of CCR2 and CCR5

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Abstract

Chemokine receptors play pivotal roles for immune cell recruitment to inflammation sites, in response to chemokine gradients (chemotaxis). The mechanisms of chemokine signaling, especially the initiation of the intracellular signaling cascade, are not well understood. We previously identified a cytoplasmic protein FROUNT, which binds to the C-terminal regions of CCR2 and CCR5 to mediate chemokine signaling. Although large amounts of purified protein are required for detailed biochemical studies and drug screening, no method to produce recombinant FROUNT has been reported. In this study, we developed a method for the production of recombinant human FROUNT. Human FROUNT was successfully expressed in Escherichia coli, as a soluble protein fused to the folding chaperone Trigger Factor, with a cold shock expression system. The purified FROUNT protein displayed CCR2 binding ability without any additional components, as demonstrated by SPR measurements. A gel filtration analysis suggested that FROUNT exists in a homo-oligomeric state. This high-yield method is cost-effective for human FROUNT production. It should be a powerful tool for further biochemical and structural studies to elucidate GPCR regulation and chemokine signaling, and also will contribute to drug development.

Introduction

G protein-coupled receptors (GPCRs) constitute the largest family of membrane proteins and mediate a multitude of cellular and physiologic responses to specific ligands [1]. Mutations in the genes encoding GPCRs are implicated in numerous diseases, and these receptors presently form the largest class of therapeutic targets [2]. Chemokine receptors play pivotal roles for immune cell recruitment to inflammation sites, in response to chemokine gradients (chemotaxis). This innate immune system is absolutely required for host defense, although when it becomes uncontrolled, it leads to inflammatory disease. Approximately 20 plasma membrane receptors have been characterized as members of the chemokine receptor family, and all of them are GPCRs [3].

Mutational analyses revealed that the cytoplasmic C-terminal domain, especially the membrane-proximal C-terminal region (Pro-C), of chemokine receptors plays an important role in chemotaxis [4], [5], [6], [7], [8], [9]. In the cases of CCR2 and CCR5, the truncation of the Pro-C also impairs the chemokine signals, without the loss of cell surface localization [4], [5]. We previously identified a 75-kDa cytoplasmic protein, FROUNT, which interacts with the Pro-C regions of CCR2 and CCR5, using a yeast two-hybrid system [10], [11]. FROUNT directly binds to activated CCR2 and CCR5 and mediates directional cell migration. Since FROUNT does not bind to the C-terminal regions of CCR1, CCR3 and CXCR4, it was suggested that FROUNT interacts specifically with CCR2 and CCR5 [11]. The mechanisms of chemokine signaling, and especially the initiation of the intracellular signaling cascade, are not well understood. Since FROUNT lacks homology with known GPCR regulators, FROUNT may mediate the chemokine signaling in a novel manner. Clarification of the function of FROUNT will provide new insights into chemokine signaling and general GPCR regulation.

CCR2 and CCR5 are involved in various diseases, including chronic inflammation, cancer progression and viral infection, and thus FROUNT is considered as a promising drug target to treat a wide range of diseases. Various reports have indicated that FROUNT could actually have effective therapeutic applications: (1) We previously reported that macrophage infiltration was inhibited by FROUNT depletion, in a mouse peritonitis model [10]. (2) Belema-Bedada et al. reported that FROUNT is required for the migration and recruitment of CCR2-expressing bone marrow-derived mesenchymal stem cells to injured heart tissue [12]. (3) Satoh et al. showed that the mRNA levels of both FROUNT and CCR2 were up-regulated in biopsy tissue samples from patients with heart failure [13]. (4) Golen et al. reported that FROUNT mediates the transendothelial migration of prostate carcinoma cells [14].

Although large amounts of purified protein are required for detailed biochemical studies and drug screening, no method to produce recombinant FROUNT has yet been reported. We report here the first successful expression and purification of human FROUNT. We expressed human FROUNT fused to Trigger factor (TF), by a cold shock expression system in Escherichia coli (E. coli). The purified FROUNT protein retained the bind ability to CCR2. A gel filtration analysis suggested that FROUNT has oligomeric properties.

Section snippets

Materials

Restriction enzymes were purchased from Toyobo Co., Ltd. PrimeStar DNA1 polymerase and pCold TF DNA were purchased from Takara Bio

Expression and purification of recombinant FROUNT

To express and isolate the human FROUNT protein, we constructed several kinds of E. coli expression plasmids encoding human FROUNT with various fusion tags, including GST, His6, and Strep (Table 1). However, all of the proteins expressed from these plasmids formed inclusion bodies in E. coli under two temperature conditions (16 and 32 °C) (Table 1). Although we tried to purify the His6-FROUNT-Strep fusion protein from the E. coli lysate, which even included a very small amount of the soluble

Conclusions

This is the first report describing the expression and purification of recombinant human FROUNT. Human FROUNT was strongly expressed, as a soluble protein fused to TF, with the cold shock expression system in E. coli. Using our method, it was purified well, with a high yield. Using this purified protein, we found that human FROUNT is able to bind to CCR2 without any additional components. We also determined that human FROUNT exists in homo-oligomeric states.

Finally, the availability of

Acknowledgments

We are grateful to Dr. Shogo Misumi for the TOF mass spectrum measurements, Dr. Kohichi Kawahara for the N-terminal amino acid sequencing, and Dr. Hiroshi Morioka and Mr. Ryu Takahashi for SPR measurements. This work was supported in part by a Grant-in-Aid from the Targeted Proteins Research Program (TPRP) from the Japan Science and Technology Agency (JST), by a Grant-in-Aid for Young Scientists (B) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT), by a

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