However, molecular relationships and signaling events following CXCL11 or CXCL12 binding to CXCR7 remain poorly defined and controversial. Several reports suggest that CXCR7, despite conserving most of the canonical GPCR features, does not activate Gi-mediated pathways that are standard for chemokine receptors and would result in DPCPX GTP hydrolysis, calcium mobilization, and chemotaxis , , . (Fsk) response and represent mean SEM.(PDF) pone.0034192.s001.pdf (120K) GUID:?C09E551D-2888-4F24-9653-6FDC452ADCB7 Figure S2: Cell surface expression of RLuc-tagged receptors. Surface manifestation of RLuc -tagged CXCR7 constructs was assessed by [125I]CXCL12 whole cell binding. Data symbolize the imply SEM of 3 experiments each performed in triplicate.(PDF) pone.0034192.s002.pdf (59K) GUID:?99772530-AD2C-4746-87BA-206748EBFC8B Number S3: CXCR7 recycles after agonist stimulation while CXCR3 downregulates upon prolonged exposure to its ligand. Receptor surface manifestation was assessed by ELISA in HEK293T cells transiently transfected with wt CXCR7 or wt CXCR3. To assess for total receptor manifestation cells were permeabilized after fixation with 0.5% NP-40. Data symbolize the imply SEM of 3 experiments each performed in triplicate.(PDF) pone.0034192.s003.pdf (77K) GUID:?8CBF1C10-97CB-4900-930D-EDC4A3801581 Number S4: CXCR7 K/A colocalization with -arrestin2. HEK293T cells were transiently transfected with CXCR7 wt or K/A (reddish channel) and -arrestin2-YFP (green channel). Cells were fixed and permeabilized prior to the immunodetection of CXCR7 with the 11G8 anti-CXCR7 antibody and an anti-mouse Alexa546-conjugated secondary antibody. Scale pub signifies 10 m.(PDF) pone.0034192.s004.pdf (1.7M) GUID:?84D0CD9D-97B2-4BD0-872C-0577F4A539F9 Figure S5: CXCR7 K/A shows increased basal interaction with -arrestin2. HEK293T cells coexpressing RLuc-tagged CXCR7 DPCPX wt or K/A mutant and YFP-tagged -arrestin2 were stimulated with 10? 8 M of CXCL12 prior to BRET measurements. Results are indicated as collapse of basal Online BRET as explained in Materials and Methods. Data symbolize the imply SEM of 3 experiments each performed in triplicate.(PDF) pone.0034192.s005.pdf (73K) GUID:?58DE63F7-F5E9-4C12-9C38-B4BE12296EB9 Table S1: Amino acid sequence of the mutated C-tails of CXCR7. Bold letters show the introduced changes from your CXCR7 original sequence. The conserved NPXXY motif is definitely underlined like a research.(DOC) pone.0034192.s006.doc (29K) GUID:?FAB44018-C636-4A22-8834-5D14841374AF Table S2: CXCL12 binding affinities for mutant CXCR7 receptors. pKd ideals were acquired by [125I]-CXCL12 homologous competition binding on membrane preparations of cells expressing CXCR7 WT or mutant receptors.(DOC) pone.0034192.s007.doc (28K) GUID:?5C0ED554-DBEB-4FDA-83C6-A903C4830759 Abstract The chemokine receptor DPCPX CXCR7 binds CXCL11 and CXCL12 with high affinity, chemokines that were previously thought to bind exclusively to CXCR4 and CXCR3, respectively. Manifestation of CXCR7 has been associated with cardiac development as well as with tumor growth and progression. Despite having all the canonical features of G protein-coupled receptors (GPCRs), the signalling pathways following CXCR7 activation remain controversial, since unlike standard chemokine receptors, CXCR7 fails to activate Gi-proteins. CXCR7 has recently been shown to interact with -arrestins and such connection has been suggested to be responsible for G protein-independent signals through ERK-1/2 phosphorylation. Transmission transduction by CXCR7 is definitely controlled in the membrane by the process of GPCR trafficking. In the present study we investigated the regulatory processes induced by CXCR7 activation as well as the molecular relationships that participate in such processes. We display that, CXCR7 internalizes and recycles back to the cell surface after agonist exposure, and that internalization isn’t just -arrestin-mediated but also dependent on the Serine/Threonine residues in the C-terminus of the receptor. Furthermore we describe, for the first time, the constitutive ubiquitination of CXCR7. Such ubiquitination is definitely a key changes responsible for the correct trafficking of CXCR7 from and to the plasma membrane. Moreover, we found that CXCR7 is definitely reversibly de-ubiquitinated upon treatment with CXCL12. Finally, we have also recognized the Lysine residues in the C-terminus of CXCR7 to be essential for receptor cell surface delivery. Collectively these data demonstrate the differential rules of CXCR7 compared to the related CXCR3 and CXCR4 receptors, and focus on the importance of understanding the molecular determinants responsible for this process. Intro CXCL12 (SDF1)-mediated effects have been classically attributed to its connection with chemokine receptor CXCR4. However, it has recently been appreciated that CXCL12 Gpc4 also binds with high affinity to chemokine receptor CXCR7 (earlier also referred to as RDC-1 or CXC-CKR2), an evolutionary conserved G DPCPX protein-coupled receptor (GPCR) , . In addition, the CXCR3-ligand CXCL11 (I-TAC) ,  has also been found to bind to CXCR7. CXCR7 plays a role in cardiac development  as well as in promoting tumor development and progression , . In fact, CXCR7 has been shown to promote the growth of tumors created from lung, breast and liver tumor cells ,  and improved manifestation of CXCR7 has been correlated with the aggressiveness of prostate malignancy , suggesting an important part for this receptor in tumor metastases and progression . More recently, it has been demonstrated that CXCR7 is also indicated in the nervous system, where it has been explained to be involved in both the development of the CNS ,  as well as with tumor malignancy . Importantly, in cortical interneurons, CXCR7 has been postulated to indirectly regulate the manifestation of CXCR4 and consequently sustain normal levels of this receptor . Similarly, in.