Research Assistant Professor University of Texas at El Paso El Paso, Texas, United States
Disclosure(s):
Colin Bill, PhD: No financial relationships to disclose
Introduction/Rationale: CCR7 is a membrane-bound G protein-coupled receptor (GPCR), and trans-locates to membrane-bound compartments in T cells. We have found that in humans and in mice signaling through CCR7/CCL19 promotes rapid receptor internalization via arrestin-3, while CCR7/CCL21 is internalized more slowly and to a lesser extent, demonstrating distinct internalization pathways for each receptor/ligand combination. In this study we determined the kinetics, rab GTPases are small GTPase molecules that regulate vesicle and cell membrane trafficking of CCR7/ligand in human T cells.
Methods: To visualize CCR7 intracellular movement in T cells we used CEM leukemia cells stably expressing rab 5, 7 or 11 fused to mCherry. Cells were fixed and endogenous CCR7 was labeled, and single cells were imaged using a Zeiss LSM 900Airyscan 2 confocal microscope.
Results: Rab proteins recruit effector proteins that control lipid membrane organization. Phosphatidylinositol-3-kinase’s which phosphorylate various phosphatidylinositol lipids on the 3’ carbon of the inositol head group that serve as anchoring molecules for rabs. PI3K Class 2 ⍺ (PI3KC2⍺) works with rab 11 to recycle GPCRs post-internalization. On analysis of untreated cells we found that CCL19 promotes rapid trafficking of CCR7 to rab 5 early endosomes followed by trafficking to rab 11 recycling endosomes for up to 2 hr. Notably, a subset of CCR7 trafficked to Rab7 lysosomes, suggesting that the receptor is being degraded after prolonged stimulation. Using 500nM wortmannin, which blocks PI3KC2alpha, we found that recycling is dependent upon class 2 PI3Ks. In contrast, for CCR7/CCL21 we found that CCR7 remained at the surface of CEM cells associated with the membrane marker ras4.
Conclusion: These results indicate that CCL19 and CCL21 are differentially trafficked in T cells with most CCR7/CCL21 remaining at the cell surface, whereas CCR7/CCL19 is both recycled and degraded. CCR7/CCL19 trafficking is rab and class 2 PI3K dependent.
