Abstract
CXCR4, the primary receptor for CXCL12, plays a critical role in the development of hematopoietic, vascular, central nervous, and immune systems by mediating directional migration of precursor cells. This mechanism promotes homing of tumor cells to metastatic sites that secrete CXCL12, and CXCR4 expression is a negative prognostic factor in acute myelogenous leukemia (AML). To elucidate mechanisms that regulate CXCR4 signaling, we used a proteomic approach to identify proteins physically associated with CXCR4. Analysis of CXCR4 immune complexes identified nucleophosmin (NPM), which was confirmed by reciprocal coimmunoprecipitation for NPM. Constitutively active CXCR4 variants bound higher levels of NPM than the wild-type receptor, which was reversed by T140, an inverse agonist. NPM binding to CXCR4 localized interactions to the C terminus and cytoplasmic loop (CL)-3, but not CL-1 or CL-2. Alanine scanning mutagenesis demonstrated that positively charged amino acids in CL-3 were critical for NPM binding. Recombinant NPM decreased GTP binding in membrane fractions after activation of CXCR4 by CXCL12. Suppression of NPM expression enhanced chemotactic responses to CXCL12, and, conversely, overexpression of a cytosolic NPM mutant reduced chemotaxis induced by CXCL12. This study provides evidence for a novel role for NPM as a negative regulator of CXCR4 signaling induced by CXCL12 that may be relevant to the biology of AML.
Footnotes
-
This work was supported by National Institutes of Health grant AI41346 and a grant from the Georgia Cancer Coalition (to S.C.P.).
-
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
-
doi:10.1124/mol.107.037119.
-
ABBREVIATIONS: GPCR, G protein-coupled receptor; HIV, human immunodeficiency virus; C-ter, C terminus; NPM, nucleophosmin; CL, cytoplasmic loop; ΔT, C terminus-truncated; GST, glutathione transferase; siRNA, small interfering RNA; CHO, Chinese hamster ovary; HEK, human embryonic kidney; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; BSA, bovine serum albumin; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; SDF, stromal cell-derived factor; LC-MS/MS, liquid chromatographytandem mass spectrometry; GTPγS, guanosine 5′-O-(3-thio)triphosphate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate; siNPM, small interfering RNA targeting nucleophosmin; WT, wild type; EGFP, enhanced green fluorescent protein.
- Received April 14, 2007.
- Accepted August 22, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|