Expression and purification of human FROUNT, a common cytosolic regulator of CCR2 and CCR5
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
References (22)
- et al.
Dissociation of chemotaxis from agonist-induced receptor internalization in a lymphocyte cell line transfected with CCR2B. Evidence that directed migration does not require rapid modulation of signaling at the receptor level
J. Biol. Chem.
(1997) - et al.
Characterization of sequence determinants within the carboxyl-terminal domain of chemokine receptor CCR5 that regulate signaling and receptor internalization
J. Biol. Chem.
(2001) - et al.
Selective modulation of wild type receptor functions by mutants of G-protein-coupled receptors
J. Biol. Chem.
(1999) - et al.
The IL sequence in the LLKIL motif in CXCR2 is required for full ligand-induced activation of Erk, Akt, and chemotaxis in HL60 cells
J. Biol. Chem.
(2006) - et al.
Ligand-induced desensitization of the human CXC chemokine receptor-2 is modulated by multiple serine residues in the carboxyl-terminal domain of the receptor
J. Biol. Chem.
(1997) - et al.
Efficient homing of multipotent adult mesenchymal stem cells depends on FROUNT-mediated clustering of CCR2
Cell Stem Cell
(2008) - et al.
A novel activator of C–C chemokine, FROUNT, is expressed with C–C chemokine receptor 2 and its ligand in failing human heart
J. Card. Fail.
(2007) - et al.
The fibronectin type III domain as a scaffold for novel binding proteins
J. Mol. Biol.
(1998) - et al.
Proteomic analysis of human brain identifies α-enolase as a novel autoantigen in Hashimoto’s encephalopathy
FEBS Lett.
(2002) - et al.
The structure and function of G-protein-coupled receptors
Nature
(2009)
Emerging concepts of guanine nucleotide-binding protein-coupled receptor (GPCR) function and implications for high throughput screening
Assay Drug Dev. Technol.
Cited by (11)
Expression of highly active chondroitin 4-O-sulfotransferase-1 in Escherichia coli by a trigger factor fusion protein expression system
2022, Process BiochemistryCitation Excerpt :Many tag fusion systems have been developed to prevent the formation of inclusion bodies. In particular, TF, which is chaperone protein, has shown several successful results in the expression of soluble proteins in E. coli [34–37]. Furthermore, TF is expressed at low temperature, which is the expression condition of active C4ST-1 in E. coli.
Preparation of low-molecular-weight citrus pectin by recombinant Bacillus subtilis pectate lyase and promotion of growth of Bifidobacterium longum
2018, Catalysis CommunicationsCitation Excerpt :Specific activity of purified rePelB measured 1006.7 ± 4.8 U/mg. The pCold TF vector system provides a cold shock technology and a folding chaperone Trigger Factor for high-level expressions of soluble recombinant proteins [18,19]. SDS-PAGE (Fig. 1A) and Western blot (Fig. 1B) analysis revealed 100.5 kDa molecular mass of rePelB.
Targeting FROUNT with disulfiram suppresses macrophage accumulation and its tumor-promoting properties
2020, Nature Communications