Ligand-dependent association of CXCR7 and -arrestin 2 elevated progressively over the full time course, reaching a maximum of 2

Ligand-dependent association of CXCR7 and -arrestin 2 elevated progressively over the full time course, reaching a maximum of 2.4-fold induction after 40 min with 1000 SBI-553 ng/mL CXCL12. more than 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 transmission 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 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 conversation. In contrast, cells expressing only CXCR7 have higher basal association with -arrestin 2 and exhibit more gradual, continuous 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 the same cell substantially decreases both the magnitude and period of CXCL12-regulated recruitment of -arrestin 2 to CXCR4. Co-expression of both receptors on the same cell only minimally alters recruitment of -arrestin 2 to CXCR7. experiments also recognized -arrestin 2 as a limiting factor in cells expressing both receptors, establishing that CXCR7 wins the competition with CXCR4 for CXCL12 and recruitment of -arrestin 2. These results reveal how SBI-553 competition for -arrestin 2 controls integrated responses to CXCL12 in cells expressing both CXCR4 and CXCR7. These results advance understanding of normal and pathologic functions of CXCL12, which is critical for developing effective strategies to target 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 [1]C[3]. The numerous signaling pathways regulated by this chemokine correspond Rabbit polyclonal to AGAP with crucial functions in development, normal physiology, and disease. Germline deletion of CXCL12 in mice is usually lethal due to abnormal development of cardiovascular, hematopoietic, and central nervous systems [4]C[6]. 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 [7], [8]. CXCL12 signals through chemokine receptors CXCR4 and CXCR7 (recently renamed ACKR3). In cells expressing only CXCR4, CXCL12 binding to CXCR4 initiates signaling pathways common 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 [9]. Conversely, CXCR7 is an atypical chemokine receptor that does not activate G proteins in response to CXCL12 [10]. CXCR7 functions in part as a chemokine decoy receptor for CXCL12, removing this chemokine from extracellular space and degrading it [11]C[13]. Functions of CXCR7 SBI-553 are enhanced by 10-fold higher binding affinity for CXCL12 relative to CXCR4 and constitutive internalization and recycling of CXCR7 to the cell membrane [12], [14]. In response to CXCL12, CXCR7 also signals through -arrestin 2 dependent pathways on endosomes [3], [15]. Cells generally 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 malignancy cells [16]. Activated macrophages increase mRNA and protein for CXCR7 while downregulating CXCR4, and platelets from patients with acute coronary artery SBI-553 disease increase CXCR7 while maintaining levels of CXCR4 [17], [18]. In addition, tumor-initiating cells from some brain malignancy cell lines may preferentially SBI-553 express CXCR4, contrasting with more differentiated malignancy cells with greater expression of CXCR7 [19]. Changes in numbers of CXCR7 versus CXCR4 receptors on cells may alter signaling pathways normally activated by CXCR4 alone, but reported effects are contradictory [20]C[22]. CXCR7 has been reported to either impair or.

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