title: Bioluminescent CXCL12 fusion protein for cellular studies of CXCR4 and CXCR7.
abstract: Chemokine CXCL12 and its two known receptors, CXCR4 and CXCR7, may play a role in diseases including tumor growth and metastasis, atherosclerosis, and HIV infection. Therefore, these molecules may be promising targets for drug development. While studies of cell signaling and high-throughput screening for drug discovery increasingly are based on luminescent assays because of their high sensitivity and signal-to-background ratio, there currently is no bioluminescent assay for chemokine[#x02013]chemokine receptor binding. To develop a bioluminescent probe for chemokine binding and cellular uptake, we fused CXCL12 to Gaussia luciferase (GL), an ATP-independent enzyme that is the smallest known luciferase. Fusing CXCL12 to Gaussia luciferase (CXCL12-GL) did not alter the bioluminescence emission spectrum and only minimally affected enzyme function under varying conditions of pH, temperature, and NaCl concentration. CXCL12-GL also activated CXCR4-dependent signaling to a comparable extent as unfused CXCL12. Using multiwell plate assays, we established that CXCR7 increases cell-associated CXCL12 to a significantly greater extent than CXCR4. We also showed that CXCL12-GL can be used to quantify inhibition of chemokine receptor binding by compounds that specifically target CXCR7. These data validate CXCL12-GL as a bioluminescent probe to investigate molecular functions of CXCR4 and CXCR7 and screen for compounds that modulate ligand-receptor binding.
authors: Center for Molecular Imaging, Department of Radiology, University of Michigan Medical School, Ann Arbor, MI, USA.
authors: Kathryn
authors: K
authors: Luker
authors: Mudit
authors: M
authors: Gupta
authors: Gary
authors: G
authors: Luker
mesh_terms: D000818:Animals; D001681:Biological Assay; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology; D002460:Cell Line; D045744:Cell Line, Tumor; D054377:Chemokine CXCL12 / Q000378:metabolism*; D033342:Copepoda / Q000201:enzymology / Q000235:genetics; D055808:Drug Discovery; D005260:Female; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007093:Imidazoles / Q000378:metabolism; D007668:Kidney / Q000166:cytology; D016086:Lentivirus / Q000235:genetics; D008156:Luciferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000235:genetics; D011719:Pyrazines / Q000378:metabolism; D054387:Receptors, CXCR / Q000378:metabolism*; D019718:Receptors, CXCR4 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D013379:Substrate Specificity; D014161:Transduction, Genetic
chemical_list: C501896:ACKR3 protein, human; D054377:Chemokine CXCL12; D007093:Imidazoles; D008164:Luminescent Proteins; D011719:Pyrazines; D054387:Receptors, CXCR; D019718:Receptors, CXCR4; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C017144:coelenterazine; D008156:Luciferases