Analysis of Clathrin-mediated Endocytosis of Epidermal Growth Factor Receptor by RNA Interference

Abstract

To identify proteins that participate in clathrin-mediated endocytosis of the epidermal growth factor receptor (EGFR), 13 endocytic proteins were depleted in HeLa cells using highly efficient small interfering RNAs that were designed using a novel selection algorithm. The effects of small interfering RNAs on the ligand-induced endocytosis of EGFR were compared with those effects on the constitutive internalization of the transferrin receptor. The knock-downs of clathrin heavy chain and dynamin produced maximal inhibitory effects on the internalization of both receptors. Depletion of α, β2, or μ2 subunits of AP-2 reduced EGF and transferrin internalization rates by 40-60%. Down-regulation of several accessory proteins individually had no effect on endocytosis but caused significant inhibition of EGF and transferrin endocytosis when the homologous proteins were depleted simultaneously. Surprisingly, knockdown of clathrin-assembly lymphoid myeloid leukemia protein, CALM, did not influence transferrin endocytosis but considerably affected EGFR internalization. Thus, CALM is the second protein besides Grb2 that appears to play a specific role in EGFR endocytosis. This study demonstrates that the efficient gene silencing by rationally designed small interfering RNA can be used as an approach to functionally analyze the entire cellular machineries, such as the clathrin-coated pits and vesicles. To identify proteins that participate in clathrin-mediated endocytosis of the epidermal growth factor receptor (EGFR), 13 endocytic proteins were depleted in HeLa cells using highly efficient small interfering RNAs that were designed using a novel selection algorithm. The effects of small interfering RNAs on the ligand-induced endocytosis of EGFR were compared with those effects on the constitutive internalization of the transferrin receptor. The knock-downs of clathrin heavy chain and dynamin produced maximal inhibitory effects on the internalization of both receptors. Depletion of α, β2, or μ2 subunits of AP-2 reduced EGF and transferrin internalization rates by 40-60%. Down-regulation of several accessory proteins individually had no effect on endocytosis but caused significant inhibition of EGF and transferrin endocytosis when the homologous proteins were depleted simultaneously. Surprisingly, knockdown of clathrin-assembly lymphoid myeloid leukemia protein, CALM, did not influence transferrin endocytosis but considerably affected EGFR internalization. Thus, CALM is the second protein besides Grb2 that appears to play a specific role in EGFR endocytosis. This study demonstrates that the efficient gene silencing by rationally designed small interfering RNA can be used as an approach to functionally analyze the entire cellular machineries, such as the clathrin-coated pits and vesicles. Clathrin-coated vesicles mediate endocytosis of many receptors, channels, transporters, and other transmembrane proteins as well as various soluble macromolecules and viruses. The main components of clathrin-coated pits and vesicles are clathrin triskelions, consisting of three heavy and three light chains (1Brodsky F.M. Chen C.Y. Knuehl C. Towler M.C. Wakeham D.E. Annu. Rev. Cell Dev. Biol. 2001; 17: 517-568Crossref PubMed Scopus (540) Google Scholar, 2Kirchhausen T. Nat. Rev. Mol. Cell Biol. 2000; 1: 187-198Crossref PubMed Scopus (422) Google Scholar). The assembly of triskelions into a cage-like lattice on the cytosolic side of the plasma membrane results in the formation of a coated pit. The major proteins that drive coated pit formation are thought to be the adaptor protein complex AP-2 (3Robinson M.S. Curr. Opin. Cell Biol. 1994; 6: 538-544Crossref PubMed Scopus (306) Google Scholar). However, it was recently suggested that CALM or its neuronal homolog AP180 is sufficient for clathrin lattice assembly on a lipid monolayer (4Ford M.G. Pearse B.M. Higgins M.K. Vallis Y. Owen D.J. Gibson A. Hopkins C.R. Evans P.R. McMahon H.T. Science. 2001; 291: 1051-1055Crossref PubMed Scopus (606) Google Scholar). AP-2 is a heterotetramer composed of 100-kDa α and β2-adaptins and 50-kDa μ2 and 17-kDa σ2 subunits. CALM and AP180 are monomeric proteins. Clathrin lattice serves as an organizing scaffold for the proteins that carry out cargo sorting, membrane invagination, vesicle scission, and uncoating. These accessory molecules include adaptor/scaffold proteins and enzymes, such as dynamin GTPase, kinases, and phosphatases (5Slepnev V.I. De Camilli P. Nat. Rev. Neurosci. 2000; 1: 161-172Crossref PubMed Scopus (426) Google Scholar). The sequence of events during coated vesicle formation is understood at the morphological level. However, the molecular mechanisms of the individual steps of clathrin-mediated endocytosis (CME) 1The abbreviations used are: CME, clathrin-mediated endocytosis; EGF, epidermal growth factor; EGFR, EGF receptor; RNAi, RNA interference; siRNA, small interfering RNA; EGF-Rh, EGF conjugated to rhodamine; Tnf, transferrin; TnfR, Tnf receptor; DMEM, Dulbecco's modified Eagle's medium; GFP, green fluorescent protein; YFP, yellow fluorescent protein. remain to be elucidated. The endocytosis of the EGF receptor (EGFR) has been one of the most popular experimental systems to study the mechanisms of CME (6Sorkin A. Von Zastrow M. Nat. Rev. Mol. Cell Biol. 2002; 3: 600-614Crossref PubMed Scopus (706) Google Scholar). Over the years a number of models have been proposed and numerous proteins have been implicated. However, these models are difficult to reconcile with each other, and the views on the mechanisms of EGFR endocytosis remain controversial. One source of the discrepancies is that the important feature of EGFR endocytosis, the saturability of the specific internalization pathway, is often overlooked (7Wiley H.S. J. Cell Biol. 1988; 107: 801-810Crossref PubMed Scopus (169) Google Scholar, 8Lund K.A. Opresko L.K. Strarbuck C. Walsh B.J. Wiley H.S. J. Biol. Chem. 1990; 265: 15713-15723Abstract Full Text PDF PubMed Google Scholar). In many types of cells, occupancy of the large number of EGFRs leads to their internalization through the slow clathrin-independent pathway because the rapid clathrin-dependent pathway becomes saturated (8Lund K.A. Opresko L.K. Strarbuck C. Walsh B.J. Wiley H.S. J. Biol. Chem. 1990; 265: 15713-15723Abstract Full Text PDF PubMed Google Scholar, 9Jiang X. Sorkin A. Traffic. 2003; 4: 529-543Crossref PubMed Scopus (107) Google Scholar). Therefore, to comparatively analyze CME of the EGFR under different experimental conditions, low EGF concentrations and cell lines expressing moderate receptor levels must be used. Recent progress in application of RNA interference (RNAi) methods in mammalian cells led to important advances in elucidation of the mechanisms of EGFR endocytosis. We first used RNAi to demonstrate an essential role of Grb2 in the CME of EGFR (10Jiang X. Huang F. Marusyk A. Sorkin A. Mol. Biol. Cell. 2003; 14: 858-870Crossref PubMed Scopus (255) Google Scholar). More recently, two groups performed analysis of EGFR endocytosis in cells depleted of clathrin and AP-2 by RNAi (11Motley A. Bright N.A. Seaman M.N. Robinson M.S. J. Cell Biol. 2003; 162: 909-918Crossref PubMed Scopus (557) Google Scholar, 12Hinrichsen L. Harborth J. Andrees L. Weber K. Ungewickell E.J. J. Biol. Chem. 2003; 278: 45160-45170Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). Both studies reported no effect of AP-2 depletion on EGFR internalization despite the dramatic reduction in the number of coated pits, while they observed contrasting effects of the clathrin knock-down on EGFR endocytosis. These studies raised the question whether EGFR utilize coated pits for internalization and proposed that, even if they do utilize coated pits, these receptors are internalized through coated pits not containing AP-2. To assess the importance of clathrin, AP-2, and other endocytic proteins for EGFR endocytosis, we used novel algorithm to generate functional siRNAs to efficiently suppress the expression of these proteins. This large scale RNAi analysis revealed that clathrin, dynamin, AP-2, Eps15/15R, and Rab5 have general roles in the CME, whereas CALM, similarly to Grb2, is required specifically for EGFR internalization. Antibodies—The monoclonal antibodies to clathrin heavy chain (TD.1) were obtained from American Type Culture Collection (Manassas, VA); EEA.1, Rab5a, and PP1 were from BD Transduction Laboratories (Los Angeles, CA); Tsg101 was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); α-actinin was from Chemicon, Inc.; and GFP was from Zymed Laboratories Inc. (South San Francisco, CA). Polyclonal antibodies to dynamin II was from Affinity Bioreagents, Inc. (Golden, CO); rabbit polyclonal antibodies Ab32 specific to β-adaptins and Ab20 to CALM were described previously (13Sorkin A. McKinsey T. Shih W. Kirchhausen T. Carpenter G. J. Biol. Chem. 1995; 270: 619-625Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 14Tebar F. Bohlander S. Sorkin A. Mol. Biol. Cell. 1999; 10: 2687-2702Crossref PubMed Scopus (243) Google Scholar). Polyclonal antibodies to clathrin light chains A and B were kindly provided by Dr. F. Brodsky (University of California at San Francisco), epsin 1 by Dr. L. Traub (University of Pittsburgh), and Eps15 Ab577 and Eps15R Ab860 by Dr. P. P. Di Fiore (European Cancer Institute, Milan, Italy). siRNA Design and Synthesis—siRNAs were selected using an advanced version of the rational design algorithm that ensures siRNA had less than 16 bp of consecutive homology to untargeted molecules. For each gene, four siRNA duplexes with the highest scores (SMARTscores) were selected, and a BLAST search was conducted (Human EST data base) to eliminate potential for off-target effects. Only those sequences with more than three mismatches against unrelated genes were selected (15Jackson A.L. Bartz S.R. Schelter J. Kobayashi S.V. Burchard J. Mao M. Li B. Cavet G. Linsley P.S. Nat. Biotechnol. 2003; 21: 635-637Crossref PubMed Scopus (1927) Google Scholar). All duplexes were synthesized in-house as 21-mers with UU overhands using a modified method of 2′-acid labile orthoester chemistry (16Scaringe S.A. Methods Enzymol. 2000; 317: 3-18Crossref PubMed Google Scholar), and the anti-sense strand was chemically phosphorylated to ensure maximized activity (17Martinez J. Patkaniowska A. Urlaub H. Luhrmann R. Tuschl T. Cell. 2002; 110: 563-574Abstract Full Text Full Text PDF PubMed Scopus (1198) Google Scholar). Grb2 siRNA was described in Ref. 10Jiang X. Huang F. Marusyk A. Sorkin A. Mol. Biol. Cell. 2003; 14: 858-870Crossref PubMed Scopus (255) Google Scholar. Sequences are presented in the “Supplemental Materials.” μ2 siRNA was provided by Dr. M. Marsh (University College, London). siRNA to α-adaptin was synthesized as described previously (11Motley A. Bright N.A. Seaman M.N. Robinson M.S. J. Cell Biol. 2003; 162: 909-918Crossref PubMed Scopus (557) Google Scholar). Cells and Transfections—HeLa cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, antibiotics, and glutamine. siRNA duplexes were resuspended in 1× siRNA Universal buffer (Dharmacon, Inc.) to 20 μm prior to transfection. HeLa cells in 12-well plates (50-60% confluent; 1 ml of DMEM/fetal bovine serum per well) were transfected twice with 4 μl of 20 μm siRNA duplex and 3 μl of LipofectAMINE 2000 reagent (Invitrogen) in 100 μl Opti-MEM medium according to manufacture's recommendations at 24-h intervals. For transfections, in which two or three siRNA duplexes were included, the amount of each duplex was decreased so that the final siRNA concentration remained constant between experiments. Cells were placed into normal culture medium 12 h prior to experiments, which were performed 3 or 4 days after the initial transfection. Transfections of α-adaptin siRNA were performed using Oligofectamin reagent (Invitrogen) as described previously (11Motley A. Bright N.A. Seaman M.N. Robinson M.S. J. Cell Biol. 2003; 162: 909-918Crossref PubMed Scopus (557) Google Scholar). Western Blot Analysis—The cells were lysed by scraping from the dish by rubber policemen into Triton X-100/glycerol solubilization buffer, and the lysates were electrophoresed as described previously (14Tebar F. Bohlander S. Sorkin A. Mol. Biol. Cell. 1999; 10: 2687-2702Crossref PubMed Scopus (243) Google Scholar). Western blot analysis was performed with several antibodies followed by detection using enhanced chemiluminescence system. Several x-ray films were analyzed to determine the linear range of the chemiluminescence signals, and the quantifications were performed using densitometry. In experiments with Eps15R-targeted siRNAs, cell lysates were subjected to immunoprecipitation with Ab860, and Eps15R was detected in immunoprecipitates by Western blot as described above. GFP Fusions—To generate the fusion of YFP with Rab5b or Rab5c (YFP-Rab5b or YFP-Rab5c), a DNA fragment encoding the full-length human Rab5b or Rab5c was obtained by PCR using Pfu polymerase (Stratagene) with a SacI restriction site introduced into the 5′ end and a KpnI site into the 3′ end and cloned into pEYFP-C1 vector (Clontech, Palo Alto, CA). GFP-CALM and were described previously (14Tebar F. Bohlander S. Sorkin A. Mol. Biol. Cell. 1999; 10: 2687-2702Crossref PubMed Scopus (243) Google Scholar, Sorkin A. J. Cell 2003; PubMed Scopus Google Scholar). of and EGF Inc.) and human were as described previously (10Jiang X. Huang F. Marusyk A. Sorkin A. Mol. Biol. Cell. 2003; 14: 858-870Crossref PubMed Scopus (255) Google Scholar). Cells grown in 12-well were with or in medium bovine serum at and the of internalized and was during linear to specific internalization constant as described previously (10Jiang X. Huang F. Marusyk A. Sorkin A. Mol. Biol. Cell. 2003; 14: 858-870Crossref PubMed Scopus (255) Google Scholar). or cells grown on were with Inc.) for at with and for as described previously (10Jiang X. Huang F. Marusyk A. Sorkin A. Mol. Biol. Cell. 2003; 14: 858-870Crossref PubMed Scopus (255) Google Scholar). A of 12 was for each experimental under through and using coated pit and proteins were to study using siRNA method on their in the CME of EGFR and of and of siRNAs were designed and synthesized using an version of the rational design algorithm A. S. W. A. Nat. Biotechnol. 2003; Scopus Google Scholar). the algorithm was by a analysis of siRNA and led to the of sequence and that are functional silencing siRNA duplexes were transfected or in into HeLa cells, and the of silencing was by Western at and 4 days after transfection. 1 that the of siRNAs and siRNA depleted coated pit proteins by of levels observed in In most the of knock-down was maximal at 3 days after siRNA transfection. CALM was days after transfection. The of siRNAs to CALM was in experiments, in which these siRNAs CALM not In three and maximal silencing effects were observed 4 days transfection. was difficult to determine the of Eps15R because Eps15R is as several which can be as L. S. G. T. Sorkin A. Di Fiore J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. S. M. M. G. Chen H. De Camilli P. Di Fiore 2002; PubMed Scopus Google Scholar). 1 the depletion of four Rab5a, and For Rab5a, than of the protein a were by four siRNA antibodies that Rab5b and Rab5c were not Rab5b and Rab5c were and in HeLa The experiments with CALM and the of fusion proteins for the of was the with Rab5a, levels of both and were reduced by than as using an against in with the of a siRNA or an siRNA was that reduced silencing by and of duplexes reduced protein levels by These results a significant those obtained using design Harborth J. Weber K. Tuschl T. 2002; PubMed Scopus Google Scholar). More the of gene knock-down with siRNAs to utilize to the functional of protein One or several siRNAs for each gene were selected to the functional effects of protein silencing on the internalization rates of two receptors, for and receptor is through clathrin-coated of EGFR through coated pits is by the Both receptors are in HeLa cells at moderate The of the rates of and were performed using experiments at to of the in the and using low concentration to of the CME pathway effects of the depletion of each of 13 proteins individually on and EGF internalization. The reduction of internalization rates of both and EGFR was caused by knock-down of clathrin heavy The of a clathrin-independent and a coated endocytosis, to a small of cells on data not of two groups reported effects of siRNA knock-down of clathrin heavy chain on EGFR internalization (11Motley A. Bright N.A. Seaman M.N. Robinson M.S. J. Cell Biol. 2003; 162: 909-918Crossref PubMed Scopus (557) Google Scholar, 12Hinrichsen L. Harborth J. Andrees L. Weber K. Ungewickell E.J. J. Biol. Chem. 2003; 278: 45160-45170Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). internalization data the essential role of clathrin in EGFR internalization (11Motley A. Bright N.A. Seaman M.N. Robinson M.S. J. Cell Biol. 2003; 162: 909-918Crossref PubMed Scopus (557) Google Scholar). The of such role reported by L. Harborth J. Andrees L. Weber K. Ungewickell E.J. J. Biol. Chem. 2003; 278: 45160-45170Abstract Full Text Full Text PDF PubMed Scopus (219) Google be by an depletion of clathrin their internalization that of the clathrin-independent endocytosis to the concentrations of EGF used. To using an endocytosis the effect of clathrin heavy chain siRNA on the of was analyzed using In cells was efficiently in after of endocytosis In in cells depleted of clathrin, no was of the of endocytosis The inhibitory effect of dynamin II depletion by siRNA on the of and the data obtained with of dynamin H. M. S. H. Methods Enzymol. 1995; PubMed Scopus (100) Google and is with the that EGFR is internalized through the pathway of for dynamin II and a number of other genes appears to be a in the of protein that must be to functional effects. For depletion of dynamin II by duplex 1 in a functional reduction of internalization than that produced by duplex or a of duplexes not These data that of siRNA is essential to the of molecules and that protein is a for the of the gene In to the essential role of clathrin heavy individual or of clathrin light chains A and B did not internalization Thus, clathrin light chains do not to play an essential role in CME under the of internalization This is with studies using interfering RNA that revealed no effect of clathrin light chain A depletion on endocytosis F. Huang A. Chen C.Y. Brodsky F.M. S. A. 2002; PubMed Scopus Google Scholar). siRNA had but specific inhibitory effect on EGFR endocytosis The endocytic rates were reduced on by in experiments with two siRNA CALM depletion did not endocytosis, protein is not an essential of the endocytic a that is in light of that of the CALM clathrin endocytosis (14Tebar F. Bohlander S. Sorkin A. Mol. Biol. Cell. 1999; 10: 2687-2702Crossref PubMed Scopus (243) Google Scholar). is that the effect by the CALM was the of interference with other clathrin proteins than of specific CALM the lattice assembly of in (4Ford M.G. Pearse B.M. Higgins M.K. Vallis Y. Owen D.J. Gibson A. Hopkins C.R. Evans P.R. McMahon H.T. Science. 2001; 291: 1051-1055Crossref PubMed Scopus (606) Google be by other proteins in siRNA studies (10Jiang X. Huang F. Marusyk A. Sorkin A. Mol. Biol. Cell. 2003; 14: 858-870Crossref PubMed Scopus (255) Google of EGFR endocytosis. in Grb2 knock-down specifically reduced the rates of EGFR internalization to the with that caused by clathrin that Grb2 is for most of the EGFR through the CME the depletion of Grb2 and CALM by siRNAs did not an effect not it is that CALM is in endocytosis. is that CALM in one of the mechanisms complex to coated The depletion of reduced the endocytosis rates of EGF and by In inhibition of EGFR endocytosis in cells transfected with siRNA to the μ2 of AP-2 was observed A. M. Marsh M. Traffic. 2003; 4: PubMed Scopus Google Thus, RNAi data are with the general role of AP-2 in This by (11Motley A. Bright N.A. Seaman M.N. Robinson M.S. J. Cell Biol. 2003; 162: 909-918Crossref PubMed Scopus (557) Google reported that siRNA knock-down of AP-2 not EGFR internalization and by and J. Cell Biol. 2003; 162: PubMed Scopus Google reported normal EGFR internalization rates in cells AP-2 was by The most of the discrepancies between these studies and results is that different methods to internalization rates were used. The studies used a of cell with at 4 followed by the at of cells with EGF at 4 of into coated pits (10Jiang X. Huang F. Marusyk A. Sorkin A. Mol. Biol. Cell. 2003; 14: 858-870Crossref PubMed Scopus (255) Google and sufficient to EGFR into coated pits in AP-2 depleted In we internalization rates by cells with the at a 4 a that is more and that in the rates of the initial steps of internalization. To for the role of AP-2 in EGFR endocytosis, α-adaptin siRNA used by (11Motley A. Bright N.A. Seaman M.N. Robinson M.S. J. Cell Biol. 2003; 162: 909-918Crossref PubMed Scopus (557) Google was in internalization that under these the depletion of α-adaptin reduced EGFR endocytosis to the with that observed in cells In was no in the internalization rates of EGFR in and cells when these rates were after 4 In data with siRNAs to AP-2 that is no significant in the AP-2 for CME of EGFR and The inhibition of endocytosis by AP-2 siRNAs in experiments be to a of AP-2 depletion that was for the functional effects as compared with of clathrin or dynamin depletion sufficient for such effects. to the experiments with clathrin heavy chain and dynamin II siRNAs, the inhibition of EGFR endocytosis by AP-2 depletion to be less than that of endocytosis. This can be to the of EGFR to be internalized through clathrin-independent in the of clathrin, AP-2, and dynamin, of siRNAs to several accessory proteins produced effects. reduction of internalization rates of both receptors was observed in cells transfected with Eps15 or Eps15R siRNAs Eps15 and Eps15R have significant sequence and protein S. Di Fiore 2002; PubMed Scopus Google Scholar). both Eps15 and Eps15R were depleted by a of two siRNAs, the rates of internalization of and were decreased by that these proteins have important but roles in of Eps15 containing for α of AP-2 endocytosis of and EGFR A. C. B. S. A. J. Cell Biol. PubMed Scopus Google Scholar, R. S. G. F. P. Di Fiore Cancer Google Scholar). However, a number of other coated pit proteins with the of AP-2 D.J. Vallis Y. Evans P.R. McMahon H.T. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, the effects of Eps15 did not a for a specific of Eps15 in endocytosis, because these effects be to interference of the Eps15 with other α Depletion of three highly homologous Rab5 in a reduction of internalization rates of and EGFR whereas siRNAs to Rab5a, and Rab5c did not internalization when transfected individually on of of Rab5 was into the specific of EGFR endocytosis A. H. C. A. J. Cell Biol. 2000; PubMed Scopus Google Scholar). RNAi experiments that is and the of a of three Rab5 is to the role of in the other knock-down of did not CME Rab5 is highly and with in the of Rab5 in CME be by the of Rab5 with other To RNAi analysis has provided in the of the individual components of clathrin-coated pits in EGFR internalization. of the of the depletion of endocytic proteins is to the of these proteins. This study demonstrates that the specific and efficient gene silencing by siRNA can be used as an approach to functionally analyze the entire cellular machineries, such as the clathrin-coated pit. We F. P. P. Di M. and L. Traub for the of the and J. for in with