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Institute for Research in Biomedicine
Istituto di Ricerca in Biomedicina

Via Vincenzo Vela 6 - CH-6500 Bellinzona
Tel. +41 91 820 0300 - Fax +41 91 820 0302 - info [at] irb [dot] usi [dot] ch

Molecular mechanisms of CXCR7 sorting and potential signaling properties

Research area: Signal Transduction

Group leaders: Marcus Thelen

Status: In progress

- CXCR7 in leukocytes. We recently confirmed the expressions and function of CXCR7 on primary human B cells. In this project we confirmed the expressions and function of CXCR7 on primary human B cells. Complementary methods were used to provide unequivocal evidence. The expression of CXCR7 on non-hematopoietic cells and neoplasms is widely accepted, however, its expression on leukocytes was challenged. To solve the dissent we analyzed the expression of CXCR7 on human B cells. We validated the efficiency of different epitope-specific monoclonal antibodies to detect CXCR7 on transfected cells and primary human B cells. The specificity of the used antibodies was further confirmed by an experimentally independent double labeling approach. To this end  CXCR7 was expressed in mammalian cells tagged at the N-terminus with an epitope suitable for covalent labeling. Moreover, examination of CXCR7-dependent scavenging of fluorescent-labeled CXCL12 revealed functional expression of the receptor on human and mouse B cells. Real-time PCR analysis of CXCR7 mRNA showed the presence of transcripts in human leukocytes. Finally two CXCR7-specific peptides were identified by mass spectrometry in immunoprecipitates from primary human B cells.

- Functional role of CXCR7 in B cells. The functional role of the atypical chemokine receptor CXCR7 was examined at late stages of B cell maturation, when B cells differentiate into antibody-secreting plasmablasts before they home to the bone marrow or to the mucosa and become long-lived plasma cells. We identified two populations of CXCR7+ cells in human tonsils, one being memory B cells the other being plasmablasts. The differential expression pattern on B cells, suggests a potential contribution of the scavenger receptor in the process of final B cell maturation. We found an inverse correlation of CXCR7 and CXCR5 cell surface levels. The findings suggest an important role of CXCR7 in regulating the migration of plasmablasts at late stages of B cell maturation.

- 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, fluorescent ligands, and fluorescent tagged markers of endosomal compartments the steps of cargo sorting are analyzed. Current investigations assign the C-terminus a critical role in receptor trafficking.

 

Ligand independent CXCR7 internalization: Tagged CXCR7 (upper panels) and CXCR4 (lower panels) were labeled at 17°C with Atto-647-conjugated Co-enzyme A using phosphopantetheinyl transferase on the surface of HeLa cells expressing fluorescent protein fusion proteins of Rab5 (green) or Rab11 (blue). Cells were shifted to 37°C for 15 min an fixed. Images were taken with a confocal laser scan microscope (Leica SP5). CXCR7 is largely internalized after 20 min associating with the early endosome marker Rab5, whereas CXCR4 does not show spontaneous internalization.