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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 4, 2004 as doi:10.1096/fj.03-0867fje. |
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,2
* Asan Institute for Life Sciences, Departments of
Internal Medicine and of
Biochemistry and Molecular Biology, University of Ulsan College of Medicine, Seoul, Korea;
Department of Cell and Developmental Biology, College of Dentistry, Seoul National University, Seoul, Korea;
Department of Biochemistry and Molecular Biology, College of Medicine, Yeungnam University, Daegu, Korea; and
Graduate School of Biotechnology, Kyung Hee University, Yongin, Korea
2Correspondence: Asan Institute for Life Sciences and Department of Internal Medicine, University of Ulsan College of Medicine, Seoul 138-736, Korea. E-mail: jesangko{at}amc.seoul.kr
SPECIFIC AIMS
LZIP, a transcription factor in various cell types, was identified as a CCR1 binding protein. The aim of this study was to confirm the interaction between CCR1 and LZIP and to characterize the regulatory function of LZIP in chemokine-induced cell migration during inflammatory cellular processes.
PRINCIPAL FINDINGS
1. Yeast two-hybrid screening
To identify new molecules that bind to CCR1 and potentially regulate their receptor function, we performed the yeast two-hybrid assay. Using the carboxyl-terminal tail of 51 amino acids of human CCR1 (CCR1-Ct tail) as bait, three positive clones were identified. All three clones were found to contain parts of LZIP (amino acids 21-129, 22-114, and 22-89) that encompass the HCF binding motif (amino acids 78-81). The LZIP (21-129) clone showed strong binding to the CCR1-Ct tail and bound neither to regions containing the second intracellular loop (2nd ICL) and parts of the flanking transmembrane domains of CCR1, CCR5, and CCR7 nor to the CCR7-Ct tail. This indicates that binding of LZIP and the CCR1-Ct tail was specific.
2. CCR1 interacts with LZIP in mammalian two-hybrid system
We used a mammalian two-hybrid system to confirm the interaction between CCR1 and LZIP. CCR1 and deletion mutants of LZIP were cloned into a mammalian two-hybrid vector and the interaction was examined. LZIP contains several distinctive regions. Besides amino-terminal and carboxyl-terminal truncated mutants, four structurally distinctive regions were targeted for mutagenesis: the 1) HCF binding region (78-81, HBM), 2) basic region (152-172), 3) leucine zipper region (178-220), 4) putative transmembrane region (229-243, TM). The mutants were transfected into CHO cells or HEK 293 cells and the protein interaction was analyzed. Results from the CAT assay indicated that CCR1 interacted with the carboxyl-terminal truncated form of LZIP (1-260) as well as full-length LZIP (1-371). The deletion mutant LZIP of the amino- and carboxyl-terminal (21-260) interacted with CCR1 almost as effectively as LZIP (1-260). However, LZIP (1-243) and LZIP (21-243) were much less effective for interaction with CCR1. The rest of the combination mutant did not interact with CCR1. These data indicate that LZIP (21-260) is an important region for interaction between LZIP and CCR1.
3. CCR1 coimmunoprecipitates with LZIP
The interaction between CCR1 and LZIP was confirmed by coimmunoprecipitation of CCR1 with a full-length LZIP or the deletion mutants of LZIP from transfected HEK 293 cells or CHO cells. Full-length CCR1 tagged with FLAG and full-length or deletion mutants of LZIP tagged with HA were transfected into HEK 293 cells or CHO cells. The cotransfected cells were immunoprecipitated with anti-HA antibody and the precipitates were analyzed by Western blot. Full-length LZIP interacted with CCR1 both in HEK 293 cells and CHO cells. We also examined the interaction between CCR1 and the deletion mutants of LZIP. CCR1 was coprecipitated with LZIP (1-260) and LZIP (21-260). Other combinations of LZIP deletion mutants, including LZIP (1-243), did not interact with CCR1. These results agree with results of the mammalian two-hybrid assay.
4. Colocalization of CCR1 and LZIP in human monocytes
We tried to visualize CCR1 and LZIP immunologically in human cells using confocal microscopy. We isolated human monocytes from heparinized whole blood of healthy donors. CCR1 was found almost exclusively in the membrane. LZIP was located predominantly in the cytosol in monocytes. Merging the two images indicates that both proteins colocalized in the membrane region. These results confirmed that CCR1 interacted with LZIP inside the cell.
5. LZIP enhances the chemotactic activity of Lkn-1
Since LZIP interacts with CCR1, we investigated whether LZIP affects the chemoattractant activities induced by CC chemokines. To examine the chemotactic activities of chemokines that bind to CCR1 in HOS/CCR1 cells, a cell migration assay was performed. Lkn-1 showed a typical bell-shaped curve for HOS/CCR1 chemoattraction, with the peak at 100 ng/mL (Fig. 1
A). MIP-1
, RANTES, and HCC-4 showed maximum activity at 1 ng/mL, 100 ng/mL, and 1000 ng/mL, respectively (Fig. 1B-D
). The chemoattractant effect of Lkn-1 increased in LZIP transfected HOS/CCR1 cells compared with vector transfected and untransfected cells (Fig. 1A
). LZIP overexpression did not affect the chemotactic activities of other chemokines that bind to CCR1 (Fig. 1B-D
). These data indicate that LZIP plays a role in regulating Lkn-1-induced cell migration by interacting with CCR1.
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We tested the effect of LZIP deletion mutants on Lkn-1-induced chemotaxis. Full-length LZIP and deletion mutant forms of LZIP were transfected into HOS/CCR1 cells and subjected to a cell migration analysis. HOS/CCR1 cells transfected with either LZIP (1-260) or LZIP (21-260) showed a comparable chemoattractant effect with LZIP full-length in HOS/CCR1 cells. The finding that the deletion above the TM region reduced activation by half implies that the upstream sequence of the TM region is required for the enhancement of Lkn-1-dependent cell migration. These results are consistent with interaction experiments between LZIP and CCR1.
We generated siRNA for LZIP (siRNA-LZIP), which can reduce the expression of LZIP to 0–50% of the endogenous level. Transfection experiments with siRNA-LZIP reduced the chemotactic activity of Lkn-1 but did not affect other CCR1 binding chemokines, indicating that LZIP is specifically involved in Lkn-1-induced chemotaxis. The activation of Lkn-1-induced chemotaxis by LZIP was confirmed in THP-1 monocytes. THP-1 cells cotransfected with CCR1 and LZIP dramatically increased chemotactic activity of Lkn-1 by 4- to 10-fold compared with control cells and cells transfected with CCR1 or LZIP alone. The interaction between LZIP and CCR1 apparently is involved in regulating Lkn-1-induced cell migration.
CONCLUSIONS AND SIGNIFICANCE
We attempted to identify new proteins that bind to CCR1 and that play critical roles in chemokine-induced signal transduction. By screening a human HeLa cDNA library using a yeast two-hybrid assay, we identified an LZIP that interacts with CCR1 and activates Lkn-1-induced chemotaxis. Biochemical analysis revealed that 1) LZIP binds to CCR1 in a mammalian two-hybrid system, 2) domain 21-260 is important for interaction between LZIP and CCR1, 3) LZIP coimmunoprecipitates with CCR1 in HEK 293 and CHO cells cotransfected with LZIP and CCR1, 4) LZIP colocalizes with CCR1 in human monocytes and transiently expressing cells, 5) LZIP enhances the chemotactic activity of Lkn-1 but does not affect the chemoattractant properties of other chemokines that bind to CCR1.
It is known that chemokines that bind to the same chemokine receptor transduce the signal through the same signaling pathway. However, our preliminary data from a chemotaxis assay using CCR1 and various CCR1-dependent chemokines showed there were differential signal transduction pathways according to the type of chemokines in spite of how theses chemokines interact with the same chemokine receptor. Therefore, although we and others have previously reported CCR1-mediated signaling, the isolation of new chemokine receptor-associated proteins provides a clue to elucidate the chemokine-induced signal transduction pathways. We report a CCR1 binding protein, LZIP, known as a transcription factor and belonging to the bZIP superfamily.
We performed a mammalian two-hybrid assay to define the functional domains of LZIP and CCR1. LZIP consists of an acidic amino-terminal activation domain (1-52), a basic DNA binding domain (152-172), a leucine zipper domain (178-220), a putative transmembrane domain (229-243), and a carboxyl-terminal proline rich domain (316-371). Various combinations of truncated mutants of LZIP were constructed and their CCR1 binding activities were individually assessed. We noted that the deletion mutant of LZIP (21-260) induced CAT reporter expression as potently as did wild-type LZIP. However, an LZIP deletion mutant (21-243) did not interact with CCR1. This deletion mutant with amino acids 21-260 contained an interacting bZIP region and a putative transmembrane region. We confirmed interaction between CCR1 and LZIP by immunoprecipitation. In agreement with results of the mammalian two-hybrid assay, LZIP (21-260) coprecipitated with CCR1 in HEK 293 cells and CHO cells cotransfected with CCR1 and LZIP. Confocal microscopy data showed that LZIP colocalized with CCR1 near the membrane region.
As a consequence of direct interaction, LZIP modulates the chemotactic activity of CC chemokine Lkn-1, leading to migration of cells. LZIP increased specifically the chemotactic activity of Lkn-1 but not the activities of other CCR1-dependent chemokines. The mechanisms underlying the involvement of LZIP in the specific activation of Lkn-1-induced chemotaxis may be interpreted in two different ways. First, as a transcription factor, LZIP expresses proteins that are required to induce cell migration in response to Lkn-1. We have reported that newly synthesized proteins are required for Lkn-1-induced cell migration. Second, LZIP participates in the regulation of signaling molecules involved in the Lkn-1 induced signal transduction pathway. We had previously reported that the pertussis toxin sensitive Gi/Go protein, PLC, and PKC are all involved in cell migration through CCR1 induced by Lkn-1. Especially among the various PKC isoforms, Lkn-1 induces cell migration by the PKC
-dependent signaling pathway. Several lines of evidence are consistent with the model that LZIP functions as a positive regulator of Lkn-1-dependent signaling through CCR1. LZIP and CCR1 colocalize predominantly in the membrane, and LZIP specifically enhances the cell migration ability of Lkn-1 but not of other CCR1-dependent chemokines. Taken together, our findings point to a specific activation of Lkn-1-induced chemotaxis by LZIP mediated through physical interaction with the chemokine receptor CCR1.
In conclusion, we report the identification and characterization of human LZIP as a CCR1 binding protein. Results from a chemotaxis assay indicate that LZIP participates in Lkn-1-induced chemotaxis signaling, possibly by regulating the signaling molecules involved in the Lkn-1-induced chemotaxis signal transduction pathway. Characterization of the regulatory role of LZIP on CCR1-mediated chemotaxis signaling should shed light on the mechanism of cell locomotion and regulation of inflammatory cellular processes.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0867fje; 
This article has been cited by other articles:
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  S.-W. Jang, Y. S. Kim, Y. R. Kim, H. J. Sung, and J. Ko Regulation of Human LZIP Expression by NF-{kappa}B and Its Involvement in Monocyte Cell Migration Induced by Lkn-1 J. Biol. Chem., April 13, 2007; 282(15): 11092 - 11100. [Abstract] [Full Text] [PDF]
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), cells transfected with vector alone (
), or cells transfected with LZIP (•) were applied to the indicated concentrations of Lkn-1 (A), MIP-1
B for chemotaxis. LZIP synergistically activates the one or several signaling molecules involved in Lkn-1-induced chemotaxis signaling. LZIP is known to cleaved by protease and the cleaved form of LZIP translocates to the nucleus where LZIP functions as a transcription factor.