A unique signal sequence of the chemokine receptor CCR7 promotes package into COPII vesicles for efficient receptor trafficking.

Chemokine receptors are considered to belong to the group of G protein-coupled receptors that use the first transmembrane domain as signal anchor sequence for membrane insertion instead of a cleavable N-terminal signal sequence. Chemokine recognition is determined by the N-termini of chemokine receptors. Here, we show that the chemokine receptor CCR7, which is essential for directed migration of adaptive immune cells, possesses a 24 amino acids long N-terminal signal sequence that is unique among chemokine receptors. This sequence is cleaved off the mature human and mouse protein. Introducing single point mutations in the hydrophobic core h-region or in the polar C-terminal segment (c-region) of the signal sequence to interfere with its cleavage retained CCR7 in the ER and prevented its surface expression. Furthermore, we demonstrate the correct topology of the 35 amino acids short extracellular N-tail of CCR7 in a deletion mutant lacking the natural signal sequence. This signal sequence deletion mutant of CCR7 is fully functional as it efficiently binds its ligand, elicits chemokine-induced calcium mobilization, and directs cell migration. However, we show that the signal sequence promotes efficient recruitment of the GPCR to ER exit sites, thereby controlling efficient ER to Golgi trafficking of CCR7 on its way to reach the plasma membrane.

Ubiquitylation of the chemokine receptor CCR7 enables efficient receptor recycling and cell migration.

The chemokine receptor CCR7 is essential for lymphocyte and dendritic cell homing to secondary lymphoid organs. Owing to the ability to induce directional migration, CCR7 and its ligands CCL19 and CCL21 are pivotal for the regulation of the immune system. Here, we identify a novel function for receptor ubiquitylation in the regulation of the trafficking process of this G-protein-coupled seven transmembrane receptor. We discovered that CCR7 is ubiquitylated in a constitutive, ligand-independent manner and that receptor ubiquitylation regulates the basal trafficking of CCR7 in the absence of chemokine. Upon CCL19 binding, we show that internalized CCR7 recycles back to the plasma membrane via the trans-Golgi network. An ubiquitylation-deficient CCR7 mutant internalized normally after ligand binding, but inefficiently recycled in immune cells and was transiently retarded in the trans-Golgi network compartment of HEK293 transfectants. Finally, we demonstrate that the lack of CCR7 ubiquitylation profoundly impairs immune cell migration. Our results provide evidence for a novel function of receptor ubiquitylation in the regulation of CCR7 recycling and immune cell migration.