Molecular mechanisms of CXCR7 sorting and potential signaling properties

Researchers:

Luise Humpert, PhD Student

Status: In progress

Decoy receptors for chemokines eliminate chemokines from the environment preventing inflammation or contributing to the resolution of inflammation. Accordingly, it was proposed that the function of this class of receptors is to regulate innate and adaptive immune responses by balancing the availability of chemokines for leukocyte trafficking. Recently it was shown that CXCR7 also controls the availability of CXCL12 during development of the central nervous system.

In general chemokine receptors were shown to interact with pertussis toxin sensitive G_i proteins. Among the various constituents of the chemokine system CXCL12 and its receptor CXCR4 possess exceptional properties. Genetic deletion of either molecule leads to a similar lethal phenotype, which is exceptional as deletion of no other receptor or chemokine is fatal within the chemokine system. The phenotype is characterized by defective lymphopoiesis and myelopoiesis, imperfect vasculature, abnormal brain and heart development leading to perinatal death. These findings led to the assumption that CXCR4 and CXCL12 represent a monogamous receptor-chemokine pair. In addition to the marked role in embryogenesis and regulation of hematopoiesis, the expression of CXCR4 strongly correlates with the metastatic potential of diverse tumor cells. Among chemokine receptors, CXCR4 has unique signaling properties capable of promoting the sustained activation of intracellular signaling cascades, which is strictly dependent on the availability of extracellular CXCL12.

Our laboratory recently described CXCR7 as novel receptor for the chemokine CXCL12. Previous phylogenetic analyses placed the receptor in direct vicinity to chemokine receptors within the rhodopsin family of GPCRs. Nevertheless, because coupling to G-proteins and typical chemokine receptor-mediated signal transduction upon ligand binding could not be demonstrated for CXCR7, the receptor should be classified as 7TMD-receptor and not as a GPCR. Despite the lack of signaling, CXCR7 appears to play a critical role in development, because targeted deletion in mice is lethal. The failure of coupling to G-proteins led to the hypothesis that the receptor may act primarily as a scavenger. Indeed, we recently provided evidence for such activity in mammalian cells. The current project focuses on investigations analyzing the mechanism of CXCR7-dependent chemokine scavenging and its role in a physiological context.

Live HEK293 cells expressing CXCR4 labeled at the N-terminus with Atto-647N (red fluorescence) were incubated with CXCL12 biotinylated at the C-terminus conjugates with Quantum dots (QD 625) (green fluorescence) and analyzed by confocal microscopy. Fluorescence resonance energy transfer (FRET) (magenta color) occurs when CXCL12 is bound to CXCR4 (yellow arrows) revealing internalized receptor ligand complexes. Brown arrows indicate endosomes where receptor and ligand colocalize, but are not bound to each other. The orange arrow indicates internalized receptor without ligand.

The project focuses on the following topics:

- Investigations of ligand-dependent and -independent receptor trafficking to elucidate the mechanism of chemokine scavenging. Intracellular trafficking of CXCR7 is remarkably different from CXCR4. The temporal and molecular mechanisms of receptor sorting are not well characterized. With the aid of receptors fluorescently tagged at their N-terminus and fluorescent ligands, the initial steps of cargo sorting will be analyzed.

- Identification of potential CXCR7-mediated atypical signal transduction leading to intracellular protein phosphorylation. Based on the notion that CXCR7-cycling is ligand enhanced and that phosphorylation events are associated with almost any receptor-mediated cell activation, ligand-induced phosphorylation events are expected, which shall allow tracking of targeted proteins and eventually the involved pathways.