The roles of annexins 1 and 2 in receptor-mediated endocytosis
Activated EGF receptors (EGFR) are sorted onto internal vesicles of multivesicular endosomes/bodies (MVBs), thus removing receptors from the recycling pathway and targeting them for degradation. The EGFR tyrosine kinase has two major substrates within MVBs, the EGFR itself and annexin 1. Annexins have been proposed to play multiple roles in membrane traffic and both annexins 1 and 2 have been localised to early transferrin positive endosomes and to MVBs. In these studies a combination of gene knockout and RNAi-induced protein depletion was used to investigate the effect of loss of annexins 1 and 2 on the formation of MVBs and on EGFR trafficking. MVBs form constitutively in unstimulated cells but EGF stimulation significantly increased both the number of MVBs formed and the number of internal vesicles per MVB. Neither annexin is required for MVB formation, but EGF- stimulated inward vesiculation, within a sub-population of EGFR-containing MVBs, is mediated through annexin 1. Consistent with a role for annexin 1 in internal vesicle formation, annexin 1 and EGFR were present on the same internal vesicles within MVBs from EGF stimulated cells, but annexin 2 was not. In annexin 1 -/- cells there was no effect on EGF degradation, but a small reduction in EGFR degradation was seen. Prolonged MAPK signalling was observed in annexin 1 -/- cells, which also exhibited enhanced EGF-stimulated cell motility. Additionally, loss of annexin 1 was found to alter the shape of mouse lung fibroblasts. EGF- stimulated phosphorylation of annexin 1 was required for annexin 1-mediated inhibition of cell motility, but not for annexin 1-mediated regulation of cell shape. Therefore, annexin 1 is involved in specific EGF-stimulated effects, including internal vesicle formation, downregulation of EGFR signalling and inhibition of cell motility.