Construction And Characterization Of A CDNA Expression Library From Human Monocyte (THP-1) And Searching For The Receptor Of Chemokine CXCL14(MIP-2γ/BRAK)

Chemokine MIP-2γ , a novel member of CXC subfamily, was cloned independently by our laboratory from a human dendritic cell (DC) cDNA library by large-scale random sequencing in 2000. (BRAK was the name given by another team and CXCL14 was the systematic name given by a international nomenclature committee, so we adopted this systematic name CXCL14 in the next texts).Studies indicated that CXCL14 was a potent chemoattractant for neutrophils, and weaker for DC, but inactive to monocytes, NK cells, and T and B lymphocytes. CXCL14 was constitutively and widely expressed in most normal tissue, but absent from many established tumor cell lines and human cancers. Up to now, the physiological roles of CXCL14 are unclear mainly because of the cognate receptor has not been identified.Chemokine receptors belong to the seven transmembrane G-protein coupled receptors (GPCRs) superfamily. GPCRs are the most common and successful target of clinically marketed drugs. There are approximate 150 so-called orphan GPCRs with no known ligand and function. Several laboratories and many companies have been devoted to deorphanize these orphan GPCRs including searching for the receptor of CXCL14. To understand the possible physiological significance of this chemokine, this study also attempted to clone the receptorfor CXCL14 using several strategies.First, we tried the GST pull-down and coimmunoprecipitation methods. These two techniques are fundamentally similar and characterize in vitro and in vivo protein interactions, respectively. In the GST pull-down method, after preparation of the plasma membranes of CXCL14 target cell KG-1 through sucrose density gradient ultracentrifugation, we incubated the plasma membrane with CXCL14-GST fusion protein which binding to the immobilized glutathione column, using GST alone protein as a negative control at the same time. After washing away the non-specific binding proteins, the column were eluted with 20mM reduced glutathione elution buffer. The elution fractions were analyzed by SDS-PAGE followed by normal and silver staining. But we hadn't observed expected extra band on the gel that associate uniquely with the CXCL14-GST fusion protein and not GST. In the coimmunoprecipitation method, the CXCL14-GST fusion protein and immobilized glutathione column were replaced by anti-human CXCL14 Antibody plus CXCL14 and protein A beads, respectively. But also no expected extra band was detected. We decided to give up these methods after trying several times.Second, we explored the strategy based on sequence homology among gene family members using PCR with degenerate primers or low stringency hybridization from libraries with degenerate oligonucleotide probes. All the known chemokine receptors in the Chemokine Receptor Family Database were multiple-aligned using ClustalX1.81.Then, according to the multiple alignment,we designed two degenerate primer pairs using the well-established CODEHOP software. Total RNA extracted from target cell KG-1 were employed as template and amplified by touch-down PCR using the degenerate primers. After agarose gel electrophoresis, the expected specific bands were recovered from the gel and cloned into the T vector. Transforming E.Coli competent cells with the recombinant plasmids and sequencing about 240 randomly picked clones. Most of the sequenced clones corresponded to the known CCR2b sequence. Several novel sequences didn't belong to the chemokine receptor family according to the BLAST analysis in GenBank. We had to abandoned again and turned to other strategies.Third, we made use of the most common strategy in cloning chemokine receptor: orphan receptor cloning strategy. Fourteen orphan GPCRs, considered as putative chemokine receptors based on sequence similarity, were cloned into the eukaryotic expression vector pDNA3.1/myc-His(-)B and cell lines stably expressed these orphan GPCRs were obtained by transfection HEK293 cells and selection with G418. Soluble CXCL14-AP fusion protein were collected from the supernatant of 293T cells transfected with CXCL14-AP fusion expression plasmid. Then we performed the binding assay with the soluble CXCL14-AP protein and the HEK293 cell lines stably expressed orphan GPCRs. To our disappointment, no specific binding was detected.At last, we chose the classical expression cloning strategy to fish out the receptor of CXCL14. Many receptors were identified by expression cloning...