Class II Phosphatidylinositol-3-kinases Promotes CCR7 Recycling to the Cell Membrane in Human T-Cells

Abstract

Abstract C-C Chemokine Receptor 7 (CCR7) and its ligands (CCL19 and CCL21) are important for immune cell migration/co-localization during the primary and subsequent immune responses. We have previously shown both in humans and in mice that signaling through CCR7/CCL19 promotes rapid receptor internalization via arrestin-3. Therefore, we questioned whether internalized CCR7/CCL19 recycles to the membrane in T-cells, and if so, what are the drivers for recycling. G protein-coupled receptors can activate phosphatidylinositol-3-kinase (PI3K) family members to regulate membrane remodeling events, which include receptor recycling to the cell surface. To better understand the role of PI3K in the recycling of CCR7, we used wortmannin at 5μM to block class I and class II PI3K’s and at 0.5μM to block class II PI3K during CCR7 recycling. We found that under both conditions, receptor recycling was reduced by 20% suggesting a role for PI3K family members in recycling of CCR7 to the cell membrane. Treatment of the cells with the class I δ specific inhibitor, GS-1101, however, did not affect recycling, suggesting that CCR7/CCL19 recycling to the cell membrane is dependent on class II PI3K. To determine the site within the cells where CCR7 was retained following inhibition of only class II PI3K, we used HEK-293 cells transfected with CCR7-eGFP and the trans-Golgi network (TGN) marker, TPST2-mCherry, ± 1μM wortmannin, which is required to inhibit class II PI3K in HEK-293 cells. We found that after receptor internalization with CCL19 and 30 minutes of recycling, CCR7-eGFP remained within the TGN in the presence of wortmannin, in contrast CCR7-eGFP, which recycled to the cell membrane in the absence of wortmannin, demonstrating a role for class II PI3K in CCR7 recycling. Supported by NIH-SC1GM111172 NIH-2U54MD007592 NSF-HRD-182675 NIH-5UL1GM118970