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Proteins in the transforming growth factor-␤ (TGF-␤) family recognize transmembrane serine/threonine kinases known as type I and type II receptors. Binding of TGF-␤ to receptors results in receptor down-regulation and signaling. Whereas previous work has focused on activities controlling TGF-␤ signaling, more recent studies have begun to address the trafficking properties of TGF-␤ receptors. In this report, it is shown that receptors undergo recycling both in the presence and absence of ligand activation, with the rates of internalization and recycling being unaffected by ligand binding. Recycling occurs as receptors are most likely internalized through clathrin-coated pits, and then returned to the plasma membrane via a rab11-dependent, rab4-independent mechanism. Together, the results suggest a mechanism wherein activated TGF-␤ receptors are directed to a distinct endocytic pathway for down-regulation and clathrin-dependent degradation after one or more rounds of recycling. INTRODUCTIONTGF-␤ is a ubiquitous 25-kDa polypeptide that regulates a variety of cellular processes, including matrix deposition, mitosis, development, differentiation, and apoptosis (Roberts, 1992;ten Dijke et al., 1996). The response to TGF-␤ treatment usually depends on the cell type involved, with effects as diverse as growth and growth inhibition (Massagué, 1996;Moses and Serra, 1996). TGF-␤ binds to singlepass transmembrane serine/threonine kinases referred to as type I and II TGF-␤ receptors (Bassing et al., 1994;ten Dijke et al., 1994). On binding of TGF-␤ to the constitutively active type II receptor (T2R), the type I receptor (T1R) is recruited and phosphorylated by T2R. The activated T1R then phosphorylates downstream signaling intermediates such as the Smad proteins, which translocate to the nucleus and function as transcriptional comodulators (Franzén et al., 1993;Macias-Silva et al., 1996;Yingling et al., 1996).Study of the endocytic response of TGF-␤ receptors to ligand has been difficult due to nonspecific TGF-␤ binding and the fact that different receptor complexes form on the cell surface (heteromers vs. homomers) and undergo distinct endocytic fates (Anders et al., 1997). To overcome these problems, our laboratory created a chimeric receptor system where the ligand binding extracellular domains of the granulocyte/macrophage-colony stimulating factor (GM-CSF) receptors were fused to the transmembrane and cytoplasmic domains of the type I and type II TGF-␤ receptors (Anders and Leof, 1996). Using a number of well-established assays for TGF-␤ action, it could be shown that TGF-␤ signaling is fully recapitulated by the chimeric system (Anders and Leof, 1996;Anders et al., 1997Anders et al., , 1998. In addition, use of the chimeric system (Anders et al., 1997) facilitated TGF-␤ endocytic assays, which showed, similar to other studies (Ehrlich et al., 2001;Yao et al., 2002), that ligand-induced internalization of TGF-␤ receptor complexes occurs through a clathrindependent process. Other reports, however, have proposed roles for...

Internalization of some plasma membrane constituents, bacterial toxins, and viruses occurs via caveolae; however, the factors that regulate caveolar internalization are still unclear. Here, we demonstrate that a brief treatment of cultured cells with natural or synthetic glycosphingolipids (GSLs) or elevation of cholesterol (either by acute treatment with m␤-cyclodextrin/cholesterol or by alteration of growth conditions) dramatically stimulates caveolar endocytosis with little or no effect on other endocytic mechanisms. These treatments also stimulated the movement of GFP-labeled vesicles in cells transfected with caveolin-1-GFP and reduced the number of surface-connected caveolae seen by electron microscopy. In contrast, overexpression of caveolin-1 decreased caveolar uptake, but treatment with GSLs reversed this effect and stimulated caveolar endocytosis. Stimulation of caveolar endocytosis did not occur using ceramide or phosphatidylcholine and was not due to GSL degradation because similar results were obtained using a nonhydrolyzable GSL analog. Stimulated caveolar endocytosis required src kinase and PKC-␣ activity as shown by i) use of pharmacological inhibitors, ii) expression of kinase inactive src or dominant negative PKC␣, and iii) stimulation of src kinase activity upon addition of GSLs or cholesterol. These results suggest that caveolar endocytosis is regulated by a balance of caveolin-1, cholesterol, and GSLs at the plasma membrane. INTRODUCTIONCaveolae are plasma membrane (PM) specializations that are rich in cholesterol, sphingolipids, and caveolin-1 (Cav1), a cholesterol-binding protein (Smart et al., 1999). By electron microscopy, they appear as flask-shaped structures at the PM and as smooth uncoated vesicles near the PM. Caveolae have been implicated in signaling, endocytosis, transcytosis, and potocytosis (for reviews see Smart et al., 1999;Fielding and Fielding, 2001;Mineo and Anderson, 2001;Carver and Schnitzer, 2003;Parton, 2003). Recently caveolae have gained attention because they have been documented to play a role in the cellular uptake and intracellular delivery of some bacterial toxins, viruses, and bacteria (Lencer et al., 1999;Shin et al., 2000;Norkin, 2001;Pelkmans et al., 2001;Richterova et al., 2001;Duncan et al., 2002;Marjomaki et al., 2002). One of the first markers to be used for caveolar endocytosis was the cholera toxin B subunit (CtxB), which binds to GM 1 ganglioside at the PM and is internalized through caveolae in some cell types (Orlandi and Fishman, 1998;Torgersen et al., 2001). SV40 virus has been shown to be internalized from the PM in small Cav1-containing vesicles (Pelkmans et al., 2001), and caveolar endocytosis of derivatized albumin has also been reported (Schnitzer et al., 1994;Shubert et al., 2001;Sharma et al., 2003;Singh et al., 2003). We previously showed that fluorescent glycosphingolipid (GSL) analogs (lactosylceramide [LacCer] and globoside) are selectively internalized via caveolae in human skin fibroblasts (HSFs) and other cell types using pathway-sp...

We recently showed that human skin fibroblasts internalize fluorescent analogues of the glycosphingolipids lactosylceramide and globoside almost exclusively by a clathrin-independent mechanism involving caveolae. In contrast, a sphingomyelin analogue is internalized approximately equally via clathrin-dependent and caveolar routes. Here, we further characterized the caveolar pathway for glycosphingolipids, showing that Golgi targeting of sphingolipids internalized via caveolae required microtubules and phosphoinositol 3-kinases and was inhibited in cells expressing dominant-negative Rab7 and Rab9 constructs. In addition, overexpression of wild-type Rab7 or Rab9 (but not Rab11) in Niemann-Pick type C (NP-C) lipid storage disease fibroblasts resulted in correction of lipid trafficking defects, including restoration of Golgi targeting of fluorescent lactosylceramide and endogenous GM 1 ganglioside, and a dramatic reduction in intracellular cholesterol stores. Our results demonstrate a role for Rab7 and Rab9 in the Golgi targeting of glycosphingolipids and suggest a new therapeutic approach for restoring normal lipid trafficking in NP-C cells.

Intracellular trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) is a focus of attention because itis defective in most patients with cystic fibrosis. ⌬F508 CFTR, which does not mature conformationally, normally does not exit the endoplasmic reticulum, but if induced to do so at reduced temperature is short-lived at the surface. We used external epitope-tagged constructs to elucidate the itinerary and kinetics of wild type and ⌬F508 CFTR in the endocytic pathway and visualized movement of CFTR from the surface to intracellular compartments. Modulation of different endocytic steps with low temperature (16°C) block, protease inhibitors, and overexpression of wild type and mutant Rab GTPases revealed that surface CFTR enters several different routes, including a Rab5-dependent initial step to early endosomes, then either Rab11-dependent recycling back to the surface or Rab7-regulated movement to late endosomes or alternatively Rab9-mediated transit to the trans-Golgi network. Without any of these modulations ⌬F508 CFTR rapidly disappears from and does not return to the cell surface, confirming that its altered structure is detected in the distal as well as proximal secretory pathway. Importantly, however, the mutant protein can be rescued at the plasma membrane by Rab11 overexpression, proteasome inhibitors, or inhibition of Rab5-dependent endocytosis.