Supplementary MaterialsFile S1: Contains the following files: Figure A: Photon flux values for (A) CXCR4-CBRN and (B) CXCR7-CBGN corresponding to Fig 3A, 3B. 20 different human cancers, as well as pathogenesis of other common diseases. CXCL12 binds two different receptors, CXCR4 and CXCR7, both of which recruit and signal through the cytosolic adapter protein -arrestin 2. Differences in CXCL12-dependent recruitment of -arrestin 2 in cells expressing one or both receptors remain poorly defined. To quantitatively investigate parameters controlling association of -arrestin 2 with CXCR4 or CXCR7 in cells co-expressing both receptors, we used a APD668 systems biology approach combining real-time, multi-spectral luciferase complementation imaging with computational modeling. Cells expressing only CXCR4 maintain low basal association with -arrestin 2, and CXCL12 induces a rapid, transient increase in this interaction. In contrast, cells expressing only CXCR7 have higher basal association with -arrestin APD668 2 and exhibit more gradual, prolonged recruitment of -arrestin 2 in response to CXCL12. We developed and fit a data-driven computational model for association of either CXCR4 or CXCR7 with -arrestin 2 in cells expressing only one type of receptor. We then experimentally validated model predictions that co-expression of CXCR4 and CXCR7 on a single cell substantially lowers both magnitude and length of CXCL12-controlled recruitment of -arrestin 2 to CXCR4. Co-expression of both receptors on a single cell only alters recruitment of -arrestin 2 to CXCR7 minimally. experiments also determined -arrestin 2 like a limiting element in cells expressing both receptors, establishing that CXCR7 wins your competition with CXCR4 for recruitment and CXCL12 of -arrestin 2. These outcomes reveal how competition for -arrestin 2 settings integrated reactions to CXCL12 in cells expressing both CXCR4 and CXCR7. These total APD668 outcomes progress knowledge of regular and pathologic features of CXCL12, which is crucial for developing effective ways of focus on these pathways therapeutically. Introduction Chemokine CXCL12 activates multiple intracellular networks, including mitogen activated protein kinases (MAPK), PI3 kinase-AKT, and JAK-Stat, to control proliferation, survival, chemotaxis, transcription, and other cellular responses C. The numerous signaling pathways regulated by this chemokine correspond with critical functions in development, normal physiology, and disease. Germline deletion of CXCL12 APD668 in mice is lethal due to abnormal development of cardiovascular, hematopoietic, and central nervous systems C. CXCL12 controls trafficking of immune cells and homing and retention of hematopoietic stem cells in bone marrow. CXCL12-dependent pathways promote growth and metastasis of more than 20 different human malignancies, and this chemokine also affects pathogenesis of other common diseases such as atherosclerosis, multiple sclerosis, rheumatoid arthritis and diabetes , . CXCL12 signals through chemokine receptors CXCR4 and CXCR7 (recently renamed ACKR3). In cells expressing only CXCR4, CXCL12 binding to CXCR4 initiates signaling pathways typical of seven transmembrane receptors, including activation of heterotrimeric G proteins and recruitment of the cytosolic adapter protein -arrestin 2. The CXCR4–arrestin 2 complex internalizes to endosomes, initiating -arrestin-dependent signaling and ultimately leading to receptor degradation . Conversely, CXCR7 is an atypical chemokine receptor that does not activate G proteins in response to CXCL12 . CXCR7 functions in part as a chemokine decoy receptor for CXCL12, removing this chemokine from extracellular space and degrading it C. Functions of CXCR7 are enhanced by 10-fold higher binding affinity for CXCL12 relative to CXCR4 and constitutive internalization and recycling of CXCR7 to the cell membrane , . In response to CXCL12, CXCR7 also signals through -arrestin 2 dependent pathways on endosomes APD668 , . Cells commonly co-express CXCR4 and CXCR7 under both normal and pathologic conditions, and studies strongly suggest that cells regulate levels of these receptors to respond to the environment and acquire new functions. For example, estrogen has been reported to increase expression of CXCR4 while reducing amounts of CXCR7 on breast Klf2 cancer cells . Activated macrophages increase mRNA and protein for CXCR7 while downregulating CXCR4, and platelets from patients with acute coronary artery disease increase CXCR7 while maintaining levels of CXCR4 , . In addition, tumor-initiating cells from some brain cancer cell.