Abstract
As an α-chemokine receptor specific for stromal-derived-factor-1 (SDF-1, also called CXCL12), C-X-C chemokine receptor type 4 (CXCR4) plays a vital role in chemotactically attracting lymphocytes during inflammation. CXCR4 also regulates HIV infection due to its role as one of the chemokine coreceptors for HIV entry into CD4+ T cells. Chemokine receptors and their signaling pathways have been shown to be regulated by the process of ubiquitination, a posttranslational modification, guided by ubiquitin E3 ligases, which covalently links ubiquitin chains to lysine residues within target substrates. Here we describe a novel mechanism regulating CXCR4 protein levels and subsequent CXCR4/CXCL12 signaling pathway through the ubiquitination and degradation of the receptor in response to ligand stimulation. We identify that an uncharacterized really interesting new gene (RING) finger ubiquitin E3 ligase, RING finger protein 113A (RNF113A), directly ubiquitinates CXCR4 in cells, leading to CXCR4 degradation, and therefore disrupts the signaling cascade. We determined that the K331 residue within CXCR4 is essential for RNF113A-mediated ubiquitin conjugation. Overexpression of RNF113A significantly reduces CXCL12-induced kinase activation in HeLa cells, whereas RNF113A knockdown enhances CXCL12-induced downstream signaling. Further, RNF113A expression and silencing directly affect cell motility in a wound healing assay. These results suggest that RNF113A plays an important role in CXCR4 signaling through the ubiquitination and degradation of CXCR4. This mechanistic study might provide new understanding of HIV immunity and neutrophil activation and motility regulated by CXCR4.
Highlights
C-X-C CHEMOKINE RECEPTOR type 4 (CXCR4) is a G proteincoupled receptor on the cellular membrane and is expressed across many tissue types
We screened a library of really interesting new gene (RING) E3 ligases to assay their activity on CXCR4 stability and observed a previously uncharacterized E3 ligase, RING finger protein 113A (RNF113A), mediates CXCR4 degradation
We recapitulated that CXCR4 protein is degraded upon CXCL12 exposure, as well as increased ubiquitin signal with pulled-down CXCR4 following CXCL12 treatment
Summary
Plasmids (pLKO.1) encoding shRNA against RNF113A were from GE Dharmacon. For plasmid overexpression in HeLa cells, plasmids were combined with XtremeGene HP kit following manufacturer’s protocol. For RNF113A silencing studies in HeLa cells, scrambled shRNA control and RNF113A shRNA were transfected into cells for 48 h using XtremeGene HP kit following the manufacturer’s protocol. Exposed cells were collected and processed for immunoblotting. Full-length CXCR4-HIS-V5 plasmid was overexpressed in HeLa cells without and with coexpression of tagless RNF113A using the above protocol. CXCR4 protein was immunoprecipitated from 1 mg HeLa cell lysate using CXCR4 antibody (rabbit) and coupled to protein A/G agarose resin. RNF113A protein was immunoprecipitated from HeLa cell lysate using. Plasmids encoding shRNA were delivered to cells using transfection protocols described above. HeLa cells were transfected with empty plasmid, RNF113A-V5, control shRNA, or shRNA against RNF113A as described above.
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