Differential PI 3-kinase dependence of early and late phases of recycling of the internalized AT1 angiotensin receptor

Agonist-induced endocytosis and processing of the G protein-coupled AT, angiotensin 11 (Ang 11) receptor (AT(1)R) was studied in HEK 293 cells expressing green fluorescent protein (GFP)- or hemagglutinin epitope-tagged forms of the receptor. After stimulation with Ang 11, the receptor and its ligand colocalized with Rab5-GFP and Rab4-GFP in early endosomes, and subsequently with Rab11-GFP in pericentriolar recycling endosomes. Inhibition of phosphaticlylinositol (PI) 3-kinase by wortmannin (WT) or LY294002 caused the formation of large endosomal vesicles of heterogeneous Rab composition, containing the ligand-receptor complex in their limiting membranes and in small associated vesicular structures. In contrast to Alexa((R))-transferrin, which was mainly found in small vesicles associated with the outside of large vesicles in WT-treated cells, rhodamine-Ang 11 was also segregated into small internal vesicles. In cells labeled with I-125-Ang 11, WT treatment did not impair the rate of receptor endocytosis, but significantly reduced the initial phase of receptor recycling without affecting its slow component. Similarly, WT inhibited the early, but not the slow, component of the recovery of AT(1)R at the cell surface after termination of Ang 11 stimulation. These data indicate that internalized AT, receptors are processed via vesicles that resemble multivesicular bodies, and recycle to the cell surface by a rapid PI 3-kinase-dependent recycling route, as well as by a slower pathway that is less sensitive to PI 3-kinase inhibitors.