Ubiquitination of CXCR7 Controls Receptor Trafficking

Abstract

The chemokine receptor CXCR7 binds CXCL11 and CXCL12 with high affinity, chemokines that were previously thought to bind exclusively to CXCR4 and CXCR3, respectively. Expression 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 typical chemokine receptors, CXCR7 fails to activate Gαi-proteins. CXCR7 has recently been shown to interact with β-arrestins and such interaction has been suggested to be responsible for G protein-independent signals through ERK-1/2 phosphorylation. Signal transduction by CXCR7 is controlled at the membrane by the process of GPCR trafficking. In the present study we investigated the regulatory processes triggered by CXCR7 activation as well as the molecular interactions that participate in such processes. We show that, CXCR7 internalizes and recycles back to the cell surface after agonist exposure, and that internalization is not only β-arrestin-mediated but also dependent on the Serine/Threonine residues at the C-terminus of the receptor. Furthermore we describe, for the first time, the constitutive ubiquitination of CXCR7. Such ubiquitination is a key modification responsible for the correct trafficking of CXCR7 from and to the plasma membrane. Moreover, we found that CXCR7 is reversibly de-ubiquitinated upon treatment with CXCL12. Finally, we have also identified the Lysine residues at the C-terminus of CXCR7 to be essential for receptor cell surface delivery. Together these data demonstrate the differential regulation of CXCR7 compared to the related CXCR3 and CXCR4 receptors, and highlight the importance of understanding the molecular determinants responsible for this process.

Rapid Uptake and Degradation of CXCL12 Depend on CXCR7 Carboxyl-terminal Serine/Threonine Residues

“…Our finding that the CXCR4 C terminus shows strong constitutive phosphorylation when fused to CXCR7 points to the possibility that the CXCR7 protein prefers a conformation that attracts G protein-coupled receptor kinases. Indeed, conversion of all C-terminal serine and threonine residues into alanines eliminates ␤-arrestin recruitment and impairs CXCR7 trafficking (37). These findings are supported by our results showing that CXCR7-mediated CXCL12 uptake and CXCL12 degradation are slower with the CXCR7-ST/A mutant.…”

“…Thus, the CXCR7 C-terminal domain accelerates not only ligand-independent receptor internalization but also ligand-independent receptor degradation. Given that CXCR7 is ubiquitinated at lysine residues in the C terminus (37) and that the rapid loss of total and surface CXCR7 receptors during cycloheximide exposure were attenuated in our CXCR7-K/R mutant, it seems likely that C-terminal ubiquitination reduces stability of the CXCR7 protein.…”

“…CXCR7 C-terminal Serine/Threonine Residues Influence the CXCL12 Scavenger Activity of CXCR7-Recently, it was reported that mutations in the CXCR7 C-terminal domain including truncation and conversion of all C-terminal lysine residues or serine/threonine residues into alanines affect CXCR7 trafficking (37,38). We therefore examined the internalization rate and scavenger activity of our CXCR7-K/R mutant and of three mutants with C-terminal ST/A conversions: CXCR7-ST/A (conversion of all C-terminal Ser and Thr residues), CXCR7-STT/A (carrying S335A, T338A, and T341A), and CXCR7-STS/A (carrying S350A, T352A, and S355A).…”

Allosteric Modulation of M1 Muscarinic Acetylcholine Receptor Internalization and Subcellular Trafficking

“…7B) suggested that some M 1 mAChRs might be targeted to the late endosomes/lysosomes. We, therefore, investigated the extent of receptor degradation after different ligand combinations using a whole cell anti-myc ELISA on permeabilized cells, which allowed us to detect total receptors (32). Although the total amount of receptors was not affected after incubation with CCh or BQCA alone followed by ligand washout, a significant decrease was observed after washout of CCh and BQCA as well as when BQCA was maintained in the recovery media (Fig.…”

Ubiquitination of G Protein-Coupled Receptors: Functional Implications and Drug Discovery

“…Ubiquitination of the platelet-activating factor receptor occurs at steady state, and it is not modulated by agonist stimulation, but seems to regulate receptor degradation after agonist stimulation (Dupré et al, 2003). In contrast, CXCR7 is basally ubiquitinated, but, upon agonist stimulation, becomes deubiquitinated in a process that requires receptor phosphorylation and ␤-arrestin recruitment (Canals et al, 2012). It is noteworthy that ubiquitination of CXCR7 is restored upon removal of ligand, resulting in recycling of internalized receptor back to the cell surface.…”