Endocytosis of GPI-linked Membrane Folate Receptor-t


GPI-linked membrane folate receptors (MFRs) have been implicated in the receptor-mediated uptake of reduced folate cofactors and folate-based chemotherapeutic drugs. We have studied the biosynthetic transport to and internalization of MFR isoform et in KB-cells. MFR-et was synthesized as a 32-kD protein and converted in a maturely glycosylated 36-38-kD protein 1 h after synthesis. 32-kD MFR-a was completely soluble in Triton X-100 at 0°C. In contrast, only 33 % of the 36-38-kD species could be solubilized at these conditions whereas complete solubilization was obtained in Triton X-100 at 37°C or in the presence of saponin at 0°C. Similar solubilization characteristics were found when MFR-et at the plasma membrane was labeled with a crosslinkable 125I-labeled photoaffinityanalog of folic acid as a ligand. Triton X-100-insoluble membrane domains containing MFR-a could be separated from soluble MFR-a on sucrose flotation gradients. Only Triton X-100 soluble MFR-a was internalized from the plasma membrane. The reduced-folate-carrier, an integral membrane protein capable of translocating (anti-)folates across membranes, was completely excluded from the Triton X-100-resistant membrane domains. Internalized MFR-a recycled slowly to the cell surface during which it remained soluble in Triton X-100 at 0°C. Using immunoelectron microscopy, we found MFR-o~ along the entire endocytic pathway: in clathrin-coated buds and vesicles, and in small and large endosomal vacuoles. In conclusion, our data indicate that a large fraction, if not all, of internalizing MFR-et bypasses caveolae. -METHYLTETRAHYDROFOLIC acid is an essential vitamin for the biosynthesis of deoxythymidylic acid, purines and the amino acids methionine and serine (Antony, 1992). For several decades the folate-metabolism has been exploited as a target for chemotherapeutic treatment of a variety of cancers using cytotoxic folate-antagonists such as methotrexate (Bertino, 1993). Transport across membranes of (anti-)folates is one of the critical determinants in their chemotherapeutic effectiveness. Two functionally different systems have been implicated in their cellular uptake: (1) the Reduced-Folate-Carrier (RFC) 1 (Sirotnak, 1985), which probably uses an anionexchange mechanism to introduce (anti-)folates into the cytosol (Henderson et al., 1986), and (2) Membrane Folate Please address all correspondence to Dr. G. J. Strous, Faculty of Medicine and Institute of Biomembranes, Department of Cell Biology, Universiteit Utrecht, AZU, room H02.314, Heidelberglaan 100, 3584' CX Utrecht, The Netherlands. Tel.: 31 30 506476. Fax: 31 30 541797. 1. Abbreviat ions used in this paper: GPI, glycosyl-phosphatidyl-inositol; IEF, isoelectric focusingi MFR, membrane folate receptor; MFR-a, membrane folate receptor-isoform ~; RFC, reduced-folate-carrier. Receptors (MFRs), which internalize (anti-)folates by a receptor-mediated process. After internalization, folates are retained in the cytoplasm by polyglutamation (McGuire et al., 1980). The transport kinetics by and affinities for (anti-)folates of both systems differ significantly (Ratnam and Freisheim, 1990; Henderson, 1990; Anthony, 1992). The RFC has a relatively high affinity for reduced folates and the antifolate methotrexate (Kin: 1-10 txM) but has a relatively poor affinity for the oxidized'folate, folic acid (Km: 200400 ~M). In contrast, MFRs have high affinities for folic acid (K~: 0.1-1 nM) and reduced folates (Kd: 1-10 nM) but relatively low affinities for methotrexate (Ka > 100 nM). Both of these transport systems can be expressed separately or complementary to each other in one cell (Jansen et al., 1989; Westerhof et al:, 1991, 1993, 1994). The function of such simultaneous expression of both systems in one cell remains unclear. Although the cell and tissue distribution of MFRs (Weitman et al., 1992; Ross et al., 1994) has been documented in gre~iter detail than the RFC (Matherly et al., 1994), both systems are constitutively expressed in many tissues and ~celi types. Recently two cDNAs have been isolated encoding a protein with RFC-activity © The Rockefeller University Press, 0021-9525/96/01/35/13 $2.00 The Joumal of Cell Biology, Volume 132, Numbers 1 & 2, January 1996 35-47 35 from mouse (Dixon et al., 1994), hamster (Williams et al., 1994), and human (Williams and Flintoff, 1995) cDNAlibraries, predicting that the RFC is a glycosylated integral membrane protein that spans the membrane 12 times. While the translocation of folates by the RFC occurs directly at the plasma membrane (Sirotnak, 1985), the uptake by MFRs is thought to occur after internalization of the receptors into the cells. Several cDNAs encoding at least three different isoforms of MFRs have been isolated (Ratnam et al., 1989; Elwood, 1989; Lacey et al., 1989; Sadasivan and Rothenberg, 1989; Brigle et al., 1991; Shen et al., 1994), and it has been well established that their products are attached to membranes by a glycosyl-phosphatidyl-inositol (GPI)-anchor (Lacey et al., 1989; Luhrs and Slomiany, 1989; Alberti et al., 1990). In general, GPIlinked proteins are internalized by cells more slowly than integral membrane receptors and possibly through different internalization pathways (Low, 1989; Lisanti et al., 1990; Bamezai et al., 1992; Keller et al., 1992; Watts and Marsh, 1992). Especially with respect to the uptake of (anti-)folates, Anderson and colleagues have shown that in MA-104 monkey kidney cells MFR-c~ binds and internalizes folates after which the receptor recycles to the cell surface. Furthermore, their study provided evidence that MFR-a works in tandem with a probenecid-sensitive carrier (Kamen et al., 1988, 1991). Electronmicroscopy in combination with these biochemical data led them to propose a model for the uptake of small molecules such as (anti-)folates, termed potocytosis (Rothberg et al., 1990a; Anderson, 1993a). In this model MFR-~ is entirely clustered in caveolae, which were originally described as small, non-clathrin-coated invaginations at the plasma membrane (Palade and Bruns, 1968) and serve to concentrate folates. After sealing of the caveolae, rapid acidification of the caveolar content would cause dissociation of folates from MFR-a after which a putative carrier protein would translocate the folates across the membrane into the cytosol. During this process caveolae remain attached to the plasma membrane. To complete the cycle the caveolae open and expose MFR-c~ to the extracellular space again. Caveolae differ from clathrin-coated buds and vesicles which pinch off from the plasma membrane after uncoating the fuse with the endosomal system. However, recently it was shown that clustering of MFR-et and other GPI-linked proteins in caveolae could be induced by specific antibodies (Mayor et al., 1994). Since the clustering of MFR-a in caveolae, as reported by Anderson and colleagues, could have been induced by antibody binding, the mechanism by which MFR-a mediates the uptake of (anti-) folates into cells remains to be established. In addition, it has been reported that internalization of MFR-a and other GPI-linked proteins proceeds via clathrin-coated or nonclathrin-coated vesicles, both capable of fusing with the entire endosomal system (Hjelle et al., 1991; Nykjzer et al., 1992; Watts and Marsh, 1992; Van Deurs et al., 1993; Sandvig and van Deurs, 1994). Caveolae are thought to exist in many different cell types (Severs, 1988; Rothberg et al., 1992). The integrity of caveolae depends on the presence of cholesterol (Rothberg et al., 1990b; Cerneus et al., 1993). Caveolae have been shown to contain multiple GPI-linked proteins (Ying et al., 1992), an IP3-sensitive Ca2+-channel (Fujimoto et al., 1992), and an ATP-sensitive Ca2÷-pump (Fujimoto, 1993). Caveolin is a major constituent of caveolae (Glenney and Soppet, 1992; Rothberg et al., 1992). VIP21, which was shown to be identical to caveolin (Glenney, 1992), has also been localized to the trans-Golgi network and has been proposed to be part of the biosynthetic apical sorting machinery in epithelial cells (Kurzchalia et al., 1992; Dupree et al., 1993). Differential solubilization of GPI-linked proteins in non-ionic detergents (for review see Low, 1989) has also been implicated in their sorting to the apical domain of the plasma membrane in epithelial cells (Brown and Rose, 1992; Cerneus et al., 1993; Garcia et al., 1993). In this model GPI-linked proteins together with glycosphingolipids form membrane domains in the trans-Golgi network, which are then targeted to the apical domain of the plasma membrane, as initially proposed by Simons and van Meer (1988). Recently, biochemical methods based on the differential solubilization characteristics in non-ionic detergents such as Triton X-100 have become available to isolate membrane domains, which are enriched in GPIlinked proteins and may represent caveolae (Cinek and Horejsi, 1992; Sargiacomo et al., 1993; Fiedler et al., 1993; Lisanti et al., 1994a; Chang et al., 1994). In addition to GPI-linked proteins isolated Triton X-100-insoluble membrane domains also contained GTP-binding proteins and protein-tyrosine kinases. This has led to the proposition of another function for caveolae, i.e., their involvement in signal transduction (Anderson, 1993a,b; Lisanti et al., 1994b). In the present study, we have used iodinated crosslinkable photoaffinity analogs of folic acid and the antifolate methotrexate to study the trafficking of functional MFR-a and RFC molecules. We have found correlations between the solubilization characteristics of GPI-linked MFR-e~ with its biosynthesis, internalization, and recycling. Our results show that MFR-a present in detergent-resistant membrane domains are excluded from internalization. Only a small amount of Triton X-100 soluble MFR-et is internalized by the cells, at least in part via clathrin-coated vesicles, and reaches the endosomal compartments. Materials and Methods Cell Culture and Biosynthetic Labeling KB nasopharyngeal carcinoma, and leukemic CCRF-CEM-7A (Jansen et al., 1990b) cells were cultured in a 5% CO2 atmosphere in RPMI 1640 without folic acid (GIBCO BRL, Gaithersburg, MD), supplemented with 10% dialyzed FCS and 1 nM of folinic acid ([6S]-5-formyltetrahydrofolate). 24 h before each experiment the folinic acid was removed from the cells by changing the medium. For metabolic labeling, cells were depleted from methionine, pulse labeled at 37°C with 3.7 MBq/ml of [35S]methionine (Tran-S-label, 185 MBq/ml, 40 TBq/mmol, ICN Biomedicals, Inc., CA) for 15 min and chased for the indicated time periods at 37°C in culture medium containing 100 ixM unlabeled methionine (Rijnboutt et al., 1992). The media were prewarmed and equilibrated in a 5% CO2-atmosphere before use. Next the cells were lysed in PBS containing 1% Triton X-100 and 1 mM PMSF at 0°C and aliquots of the lysates were incubated at 37 or 0°C in the absence or presence of 0.2% saponin. Detergent-insoluble material was subsequently removed by centrifugation at 10,000 g at 4°C. Finally, the lysates were immunoprecipitated for MFR-a and immunoprecipitations were analyzed by separation on SDS-PAGE. Quantitation of the gels was performed using a Phospho-Imager (Molecular Dynamics, Sunnyvale, CA). The Journal of Cell Biology, Volume 132, 1996 36 Synthesis of Pte-ASA-Lys N~-Pteroyl-N~(4'-azidosalicyl)-L-Lysine was prepared by the reaction of N~-[Nl°-(trifluoroacetyl)pteroyl]-L-Lysine (McAlinden et al., 1991) and 4-azidosalicylic acid succinimyl ester in a molar ratio of 1:1.5 at 16°C in subdued light (Price et al., 1986). The trifluoroacetyl group was removed concurrently by the addition of 40 mM Li2CO 3 solution. The resulting mixture was acidified using 10% aqueous citric acid and the resulting solid was isolated after lyophylization. The pellet was washed successively with water, ethanol, and diethylether. The fine yellow powder thus obtained was ~70% pure by thin layer chromatography (Rf = 0,15) on silica (Kodak 13181) using a mixture of isopropanol, methanol, and concentrated NH4OH of 3:1:1. A single slower moving impurity (Rf = 0.06) was detected which reacted positively with ninhydrine and was assumed to be N"pteroyl-L-Lysine based upon its chromatographic retention time. This material was employed in the subsequent iodination step without further purification. The affinity for Pte-ASA-Lys is similar as for folic acid as demonstrated by equimolar competition for MFR-a of [3H]folic acid binding by Pte-ASA-Lys and Pte[125I]ASA-Lys binding by folic acid (results

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@inproceedings{Rijnboutt2002EndocytosisOG, title={Endocytosis of GPI-linked Membrane Folate Receptor-t}, author={Simon Rijnboutt and Gerrit Jansen and George Posthuma and John B. Hynes and tl and Jan H. Schornagel and G. J. A. M. Strous}, year={2002} }