Endocytosed Transferrin Receptors Recycle via Distinct Dynamin and Phosphatidylinositol 3-Kinase-dependent Pathways

Abstract

Recycling of endocytosed membrane proteins involves passage through early endosomes and recycling endosomes. Previously, we demonstrated a role for clathrin-coated vesicles in transferrin receptor recycling. These clathrin-coated vesicles are formed from recycling endosomes in a process that was inhibited in dynamin-1 G273D -overexpressing cells. Here we show a second transferrin recycling pathway, which requires phosphatidylinositol 3-kinase activity. Two unrelated phosphatidylinositol 3-kinase inhibitors, LY294002 and wortmannin, retained endocytosed transferrin in early endosomes but did not affect transfer through recycling endosomes. The inhibitory effects of LY294002 and dynamin-1 G273D on transferrin recycling were additive. In combination with brefeldin A, a drug that prevents the formation of clathrin-coated buds at recycling endosomes, LY294002 inhibited transferrin recycling synergistically. Collectively, these data indicate two distinct recycling pathways. One pathway involves transfer from early endosomes to recycling endosomes, from where clathrin/dynamin-coated vesicles provide for further transport, whereas the other route bypasses recycling endosomes and requires phosphatidylinositol 3-kinase activity.

Inhibitors of phosphoinositide 3-kinase cause defects in the postendocytic sorting of β2-adrenergic receptors

“…In previously published studies, treatment of cells with wortmannin or LY294002 reduced transferrin recycling rates [14,15,22], or had minimal effects [13,23], while the recycling kinetics of AT1 angiotensin receptors were reduced [18]. In contrast, LY294002 had no measurable effect on the recycling kinetics (k r ) of β 2 ARs (Fig.…”

“…Inhibition of PI 3-kinase reduces the rate of transferrin recycling in some studies [22], and there is overlap in the endosome compartment between internalized β 2 ARs and transferrin [7]. We thus determined whether the extent of transferrin and β 2 AR recycling were similarly inhibited by LY294002 in 12β6 cells under our experimental conditions.…”

Constitutive Endocytic Cycle of the CB1 Cannabinoid Receptor

“…5C). In addition, other compounds that have been shown to inhibit recycling of the TfR receptor (28,29) and of the AT 1A angiotensin II receptor (13), the phosphoinositide 3-kinase inhibitors wortmannin and LY294002, and brefeldin A also inhibit CB1R externalization induced by AM281 (data not shown). These compounds have a broad range of cellular effects; they unravel the constitutive endocytosis of CB1R and indicate that AM281 may act through constitutive recycling but do not allow unambiguous identification of the intracellular pathways involved.…”

Elucidation of intracellular recycling pathways leading to exocytosis of the Fc receptor, FcRn, by using multifocal plane microscopy

“…As a consequence, there is uncertainty concerning the nature of the processes that lead to exocytic release (12)(13)(14)(15). For example, the origin of the exocytosing vesicles or tubules is typically difficult to determine, and it therefore remains a matter of controversy as to whether recycling from sorting endosomes involves direct fusion of endosomes or some vesicular/tubular intermediates (16). Resolving such issues is of central importance to understanding intracellular trafficking.…”