Key points are not available for this paper at this time.
A chimera of the epidermal growth factor receptor (EGFR) and green fluorescent protein (GFP) has been engineered by fusing GFP to the carboxyl terminus of EGFR. Data are provided to demonstrate that the GFP moiety does not affect the expected functioning of EGFR. EGFR-GFP becomes phosphorylated at tyrosine residues in response to EGF and is capable of phosphorylating endogenous substrates and initiating signaling cascades. EGF-dependent association of the chimeric receptor with the clathrin adaptor protein AP-2, involved in endocytosis, and with Shc adaptor protein, which binds in close proximity to the fusion point, is not affected by the GFP moiety. Receptor down-regulation and internalization occur at rates similar to those in cells expressing wild-type EGFR. Western blot analysis reveals that lysosomal degradation of EGFR-GFP proceeds from the extracellular domain and that GFP is not preferentially cleaved. Time-dependent co-localization of EGFR-GFP and Texas Red-conjugated EGF in living cells using digital deconvolution microscopy demonstrates the trafficking of ligand-receptor complexes through the early and multivesicular endosomes followed by segregation of the ligand and receptor at the late stages of endocytosis. Time-lapse optical analysis of the early stages of endocytosis reveals localization of EGFR-GFP in the tubular-vesicular endosomal compartments. Rapid dynamics of membrane movement and fusion within these compartments were observed. This approach and the fidelity of the biochemical properties of the EGFR-GFP demonstrate that real-time visualization of trafficking and protein interactions of tyrosine kinase receptors in the presence or absence of the ligand are feasible. A chimera of the epidermal growth factor receptor (EGFR) and green fluorescent protein (GFP) has been engineered by fusing GFP to the carboxyl terminus of EGFR. Data are provided to demonstrate that the GFP moiety does not affect the expected functioning of EGFR. EGFR-GFP becomes phosphorylated at tyrosine residues in response to EGF and is capable of phosphorylating endogenous substrates and initiating signaling cascades. EGF-dependent association of the chimeric receptor with the clathrin adaptor protein AP-2, involved in endocytosis, and with Shc adaptor protein, which binds in close proximity to the fusion point, is not affected by the GFP moiety. Receptor down-regulation and internalization occur at rates similar to those in cells expressing wild-type EGFR. Western blot analysis reveals that lysosomal degradation of EGFR-GFP proceeds from the extracellular domain and that GFP is not preferentially cleaved. Time-dependent co-localization of EGFR-GFP and Texas Red-conjugated EGF in living cells using digital deconvolution microscopy demonstrates the trafficking of ligand-receptor complexes through the early and multivesicular endosomes followed by segregation of the ligand and receptor at the late stages of endocytosis. Time-lapse optical analysis of the early stages of endocytosis reveals localization of EGFR-GFP in the tubular-vesicular endosomal compartments. Rapid dynamics of membrane movement and fusion within these compartments were observed. This approach and the fidelity of the biochemical properties of the EGFR-GFP demonstrate that real-time visualization of trafficking and protein interactions of tyrosine kinase receptors in the presence or absence of the ligand are feasible. The endocytosis of epidermal growth factor receptor (EGFR) 1The abbreviations used are: EGFR, EGF receptor; GFP, green fluorescent protein; wt, wild-type; EGF, epidermal growth factor; EGF-TR, Texas Red-conjugated EGF; AP-2, clathrin adaptor protein complex; MVE, multi-vesicular endosome; DMEM, Dulbecco's modified Eagle's media; TGH, Triton X-100-glycerol-Hepes buffer; PAE, porcine aortic endothelial; Ab, antibody; PAGE, polyacrylamide gel electrophoresis; MAP, mitogen-activated protein. has served as a model to study the ligand-induced receptor-mediated endocytosis for many years. It is well established that EGF binding to the surface receptors results in down-regulation of EGFR (1Carpenter G. Cohen S. J. Cell Biol. 1976; 71: 159-171Crossref PubMed Scopus (863) Google Scholar, 2Beguinot L. Lyall R.M. Willingham M.C. Pastan I. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 2384-2388Crossref PubMed Scopus (225) Google Scholar). This down-regulation is attributed to the rapid internalization of the activated receptors via clathrin-coated pits followed by the efficient sorting of the internalized receptors to the lysosome degradation pathway (reviewed in Ref. 3Sorkin A. Waters C.M. BioEssays. 1993; 15: 375-382Crossref PubMed Scopus (233) Google Scholar). The mechanism of receptor recruitment into plasma membrane clathrin-coated pits is not well understood. Although EGF-dependent interaction of EGFR with clathrin adaptor complex AP-2 has been demonstrated by several techniques (4Sorkin A. Carpenter G. Science. 1993; 261: 612-615Crossref PubMed Scopus (213) Google Scholar, 5Boll W. Gallusser A. Kirchhausen T. Curr. Biol. 1995; 5: 1168-1178Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 6Nesterov A. Kurten R.C. Gill G.N. J. Biol. Chem. 1995; 270: 6320-6327Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), the functional significance of this interaction is not formally proven (7Nesterov A. Wiley H.S. Gill G.N. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8719-8723Crossref PubMed Scopus (75) Google Scholar, 8Sorkin A. Mazzotti M. Sorkina T. Scotto L. Beguinot L. J. Biol. Chem. 1996; 271: 13377-13384Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). In addition, EGFR can be endocytosed via clathrin-independent pathway. The protein coats involved in this route are not known. Both pathways lead to the same early endosomal compartment (9Hopkins C.R. Miller K. Beardmore J.M. J. Cell Sci. 1985; 3 (suppl.): 173-186Crossref Google Scholar). These endosomes consist of tubular-vesicular membrane networks where EGF·EGFR complexes are seen co-localized with recycling receptors, for instance, transferrin receptors (10Miller K. Beardmore J. Kanety H. Schlessinger J. Hopkins C.R. J. Cell Biol. 1986; 102: 500-509Crossref PubMed Scopus (109) Google Scholar, 11Hopkins C.R. Gibson A. Shipman M. Miller K. Nature. 1990; 346: 335-339Crossref PubMed Scopus (316) Google Scholar). Both types of receptors are rapidly recycled back to the cell surface from early endosomes (12Hopkins C.R. Trowbridge I.S. J. Cell Biol. 1983; 97: 508-521Crossref PubMed Scopus (448) Google Scholar, 13Sorkin A. Krolenko S. Kudrjavtceva N. Lazebnik J. Teslensko L. Soderquist A.M. Nikolsky N. J. Cell Biol. 1991; 112: 55-63Crossref PubMed Scopus (91) Google Scholar). However, during continuous endocytosis EGFR become segregated in the vesicular parts of endosomes and subsequently concentrated in multivesicular endosomes (MVE) (12Hopkins C.R. Trowbridge I.S. J. Cell Biol. 1983; 97: 508-521Crossref PubMed Scopus (448) Google Scholar, 14Felder S. Miller K. Moehren G. Ullrich A. Schlessinger J. Hopkins C.R. Cell. 1990; 61: 623-634Abstract Full Text PDF PubMed Scopus (352) Google Scholar). Once incorporated into internal membranes of MVE, EGFRs are incapable of recycling. MVE can directly fuse with lysosomes (15Futter C.E. Pearse A. Hewlett L.J. Hopkins C.R. J. Cell Biol. 1996; 132: 1011-1024Crossref PubMed Scopus (436) Google Scholar), which ultimately leads to the proteolytic degradation of EGFR. To date, visualization of EGFR endocytosis has been largely limited to immunocytochemical analyses on chemically fixed cells. Fixation and following sample preparation for light and electron microscopy analysis often lead to dramatic changes in the morphology of the endosomal compartments and may also cause the artifactual re-distribution of the proteins. Fluorescence microscopy and video recording of living cells using fluorescent conjugates of EGF or antibodies to EGFR have been used to follow the passage of the EGFR through the endosomal compartments (11Hopkins C.R. Gibson A. Shipman M. Miller K. Nature. 1990; 346: 335-339Crossref PubMed Scopus (316) Google Scholar, 16Schlessinger J. Shechter Y. Willingham M.C. Pastan I. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 2659-2663Crossref PubMed Scopus (458) Google Scholar). However, this approach has major limitations, including non-stoichiometric labeling of the receptors, possible modification of the receptor behavior, partial dissociation of ligand-receptor complexes in acidic organelles and sorting of ligand and receptors to different compartments, and an inability to label intracellular receptors in non-permeabilized cells. GFP from jellyfish Aequorea victoria has been utilized as a reporter molecule in the fluorescent localization of several proteins (17Chalfie M. Tu Y. Euskirchen G. Ward W.W. Prasher D.C. Science. 1994; 263: 802-805Crossref PubMed Scopus (5508) Google Scholar, 18Barak L.S. Ferguson S.S.G. Zhang J. Martenson C. Meyer T. Caron M.G. Mol. Pharmacol. 1997; 51: 177-184Crossref PubMed Scopus (201) Google Scholar, 19Kallal L. Gagnon A.W. Penn R.B. Benovic J.L. J. Biol. Chem. 1998; 273: 322-328Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). GFP stoichiometrically labels as a fusion protein when integrated into the cDNA and expressed, and it can be excited by blue light and efficiently registered as green fluorescence. However, it an for a functioning EGFR-GFP the preparation and the of biochemical properties of the functional EGFR chimera with GFP that is to the carboxyl terminus of the The of the chimera that EGFR-GFP and receptor tyrosine be in for optical of biochemical and to growth factor receptor signaling and EGF from EGF using modified as A. Waters C.M. Carpenter G. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). The of EGF with Texas using Texas to the The from Texas by gel The binding of to the cells by an of EGF and is antibodies and EGFR were a from G. Carpenter to the intracellular domain of EGFR a of L. Beguinot cells were from from to Shc and the with were from kinase from and antibodies to GFP were A green of GFP, GFP to the carboxyl terminus of EGFR by the cDNA of EGFR including and from W. C. Gill G.N. J. Cell. 1985; PubMed Scopus Google by and and into and of This a EGFR that is not to GFP, To the EGFR-GFP this and to a the to residues of the and the a to and by the back and an engineered This with to a of that into to from of and porcine aortic cells from L. were used for and cells were in Dulbecco's modified Eagle's and cells in and in a at cells were in or with were with or using the were used for with cells. of cells were by with and a of fluorescent cells by The of cells and used for as cells expressing wild-type EGFR and have been A. Mazzotti M. Sorkina T. Scotto L. Beguinot L. J. Biol. Chem. 1996; 271: 13377-13384Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, Sorkina T. A. M. Kirchhausen T. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). of expressing EGFR-GFP were established by cell into cells expressing or EGFR-GFP were with and established by limited and cell were to in by the of the The cells were were with binding or and or not with EGF in binding for the at and with were by with a into of Triton with In to were at for and with and at for were through of as and the proteins were to were with and antibodies which were with antibodies or and and as in the EGFR-GFP cells were in and with in were on in binding and for in binding at were in binding and with or EGF in binding for the cells were in and the were at were cells were and into and for as The were at for the were and EGFR with of for 3 at followed by for at The were and with with and The were with to and through an polyacrylamide The gel by a and to of EGFR proteins on expressing and EGFR-GFP were at in and were in with were in binding and with or EGF in binding for at were in as The were at for at and the were into with or for 3 at and of protein were with with and The to and Western blot analysis were as To cells in were with in binding at for The of to receptors cell and to of the internalization the the and the were rapidly with to The cells were for with at The with in the same and used to the of the cells were in to the internalized The of The of the of this for the for binding for in the presence of of EGF and not of the To EGFR cells in were for with or EGF in binding at and with DMEM, and EGF by with for and followed by with The not affect the binding properties of EGFR. The of binding on the cell surface by the cells with at for on were in binding and with were in and fixed with for at were with Triton for 3 were in the same in which Triton at for with the for and with with Texas The and were by at for The cells were in A with and the for Texas or Texas were used for visualization and recording the cells expressing EGFR-GFP were in and cells were with binding and the the and the through the GFP or Texas to the and of The cells were in at and on a for were at using a with and by The of light to of by deconvolution on To rapid dynamics of EGFR-GFP endocytosis, EGF to the cell and GFP were during at on a To EGFR, the GFP to the carboxyl terminus of EGFR terminus is a domain that and tyrosine in response to EGF and several involved in receptor signaling and a protein this domain may affect EGFR the properties of the chimeric receptor be to in optical microscopy EGFR-GFP has been and in several cell that or endogenous EGFR. Western blot analysis of the of EGFR-GFP in cells that have been used in several of EGFR. were or not with EGF, and the were with antibodies for EGFR or GFP A Both antibodies the for the chimera does not EGFR at and not GFP at not these the of with EGF, of EGFR-GFP at of and in and To the are the degradation or receptors, cells expressing EGFR-GFP were with EGF in the presence or absence of a that the lysosomal degradation of EGFR. in in the absence of EGF-dependent degradation of the receptor with the of and In degradation as demonstrated by the in of receptor and by the absence of degradation of EGFR-GFP with is the carboxyl the degradation of EGFR-GFP the extracellular domain of the The of degradation by lysosomal of the EGFR as a of ligand binding to receptor and tyrosine and is also by a gel of the receptor to an in EGF binding results in of of EGFR-GFP receptor The EGFR-GFP for at which by a to the is observed. This is by down-regulation of the demonstrates rapid tyrosine of EGFR-GFP and a of proteins EGF The of of proteins in response to EGF similar to that in cells expressing wild-type EGFR and were when of cells expressing EGFR-GFP and were The of EGFR-GFP and is the of the with the of 3 These the signaling of of kinase is a of Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). To EGFR-GFP is capable of kinase of cells expressing EGFR-GFP were with the antibodies the activated 3 demonstrates EGF-dependent of which to a within The and of kinase were the same in cells expressing EGFR-GFP and wild-type EGFR not In cells with kinase during at which the of EGFR-GFP 3 in and 3 that the chimeric EGFR-GFP functional The following receptor are by interactions of the receptor carboxyl terminus with several proteins. instance, the Shc adaptor protein and is a major in involved in signaling of the EGFR G. L. J. G. N. T. Cell. Full Text PDF PubMed Scopus Google Scholar). The binding of Shc to EGFR of and the fusion in the EGFR-GFP chimera C. Beguinot L. Carpenter G. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). of AP-2 to is in the of receptor endocytosis A. Mazzotti M. Sorkina T. Scotto L. Beguinot L. J. Biol. Chem. 1996; 271: 13377-13384Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The of EGFR-GFP to with Shc and AP-2 in cells using Western blot analysis of receptor from and cells demonstrate similar of EGF-dependent association of a of AP-2 and Shc and by not These the similar biochemical properties of the chimeric and EGFR and with and 3 demonstrate that the GFP moiety does not affect the of EGFR. The in the of the receptor is a of the of ligand of the EGFR and receptor tyrosine A demonstrates EGF-dependent down-regulation of the receptor in cells expressing different of or EGFR-GFP and in cells. In cells the of at the surface in cells expressing EGFR-GFP or is at similar rates in the presence of when EGFR-GFP or were in EGF not cause of the surface endogenous receptors in these cells are rapidly To the degradation of the receptor protein, cells expressing EGFR-GFP were with and for to with were from cell with in EGF rapid degradation of EGFR-GFP with a of This is in close with the for in cells A. Mazzotti M. Sorkina T. Scotto L. Beguinot L. J. Biol. Chem. 1996; 271: 13377-13384Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, A. Waters C.M. Carpenter G. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). The of partial of EGFR-GFP and were not to a of these by the to the extracellular domain of EGFR. The rates of EGFR down-regulation and degradation are by the rates of several trafficking including and lysosomal similar of down-regulation of EGFR-GFP and that the occur at similar for receptor To directly the rates of internalization of and EGFR-GFP via the of by cells to or of at the internalization different PAE, and cells expressing EGFR-GFP or cells expressing or EGFR-GFP at similar which to to of the internalization pathway. The of EGFR is well in and these cells have used for of EGFR-GFP and However, cells endogenous receptors and that with the visualization of have also to EGFR-GFP in a cell porcine aortic in which the endogenous EGFR is by biochemical cells are to and have a morphology that is for optical These cells were used for of growth factor receptors A. L. J. Cell Biol. 1991; 112: PubMed Scopus Google Scholar), and the are well in this cell The a the of internalization of expressing and and that EGFR-GFP and In internalization at a similar in cells expressing or EGFR-GFP of to of receptor The rates of EGF internalization and down-regulation not in cells are well within the of these rates in cell These results the of cells expressing EGFR-GFP in of endocytosis of the EGFR. when the internalization of in cells that were with or the rates of endocytosis were In the internalization of EGF in cells that endogenous receptors cell This is with an down-regulation of EGFR in these cells the in cells has a for EGFR and is by the of EGFR of by The endocytosis of EGFR-GFP in cells can be by of GFP localization in the cells with EGF for at and is in The of EGFR-GFP is seen at the and in the of the which to endosomal compartments. GFP is also in the which is to endosomal as well as The of of the cells with is to that of the of is to of the in This the Western blot analysis that of EGFR or GFP are in cells. cells expressing a similar of EGFR green is the of the of GFP in The of of EGFR-GFP that this chimera is an to study EGFR trafficking using optical microscopy of living cells. In following the endocytosis of EGFR-GFP in cells. that be to the localization of the EGF, and the EGFR. were to at and the of of green and in living cells using The binding of and on the cell surface is seen of were to for to and at in binding The of GFP and Texas were following and of continuous endocytosis at in and EGFR-GFP are seen co-localized in the same vesicular of The endosomal compartments were rapidly and often a complex morphology of a vesicular and In addition, GFP at the of the cell and of GFP in the are which to the surface receptors and receptors on the to the the late stages of intracellular EGFR-GFP and co-localized in the However, a of Texas is with the vesicular compartments that not It is possible that at the late stages of endocytosis from the EGFR-GFP and is in is that the complex is to lysosomes where GFP is Texas In Texas in cells that were to for and were in not real-time microscopy of the rapid dynamics of EGFR-GFP trafficking during the early stages of receptor endocytosis. cells with EGF were by digital expected EGFR-GFP is seen in that are seen to rapidly often a In addition, the presence of EGFR-GFP in the endosomal compartments of the vesicular Ref. 11Hopkins C.R. Gibson A. Shipman M. Miller K. Nature. 1990; 346: 335-339Crossref PubMed Scopus (316) Google and is observed. several from a of a cell the and the EGF of these demonstrates the of a tubular-vesicular and the visualization of a fusion The in a of a that a does not fuse with it and the the the compartment and with it The of including the and by a is seen in In to this fusion the has in to of a and not within the are also and during the real-time microscopy of living cells visualization of the rapid dynamics of EGFR-GFP within the tubular-vesicular endosomal networks that are not in chemically fixed cells. in the of proteins with GFP the for real-time optical analysis of protein trafficking in cells. the of the GFP chimera of a receptor tyrosine in which the GFP moiety is to the carboxyl terminus of the The fusion of GFP to the of EGFR be expected to with the receptor the domain kinase and receptor interactions with proteins. the of the GFP at the terminus the and of EGFR and ligand The of and the in using it for and for to fluorescent protein of fusion at the carboxyl of the EGFR-GFP chimera that it in and biochemical properties when with EGFR-GFP is activated by EGF as by receptor tyrosine kinase in and of kinase pathway. EGF may and that the analyses may a in of these pathways in cells. of major in and W. L. M.G. Gill G.N. J. Biol. Chem. 263: Full Text PDF PubMed Google Scholar), binding of Shc to these is affected by the fusion of GFP in close proximity to these the of a protein does not of the of the receptor The of internalization and trafficking of EGFR-GFP to be similar to that of EGFR. In or the chimera at a similar to that for in these cells and for endogenous receptors in cells. In cells not have the to rapidly and EGFR-GFP or when of EGFR are activated by of These that the of EGFR can with activated EGFR for internalization to the of the pathway and that the that the of the EGFR internalization are the of partial of the extracellular domain of EGFR-GFP be in cells of EGFR have not been A. Waters C.M. Carpenter G. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar, C.M. Carpenter G. J. Cell 1984; PubMed Scopus Google Scholar, H.S. M.G. Gill G.N. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). It is possible that GFP the rapid degradation of the receptor domain to which the of partial degradation to to The are not by the antibodies to the EGFR extracellular domain and A. Waters C.M. Carpenter G. J. Biol. Chem. 1991; Full Text PDF PubMed Google and C.M. Carpenter G. J. Cell 1984; PubMed Scopus Google Scholar). the of EGFR by antibodies on the of the carboxyl In the of the chimera by is and does not on the of the EGFR-GFP be for biochemical analyses of the receptor receptor EGF-TR, and antibodies have been used to follow the endocytosis of EGFR in living cells (11Hopkins C.R. Gibson A. Shipman M. Miller K. Nature. 1990; 346: 335-339Crossref PubMed Scopus (316) Google Scholar, 16Schlessinger J. Shechter Y. Willingham M.C. Pastan I. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 2659-2663Crossref PubMed Scopus (458) Google Scholar). of the GFP to follow the localization of EGF and EGFR in living It that and EGFR-GFP are in the same compartments during the early stages of endocytosis become segregated at The co-localization of and EGFR-GFP when not These are with the that the of complexes in early endosomes and MVE A. L. Nikolsky N. Cell PubMed Scopus Google Scholar, J. Cell Biol. PubMed Scopus Google Scholar, A. Carpenter G. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). The segregation of from the receptor during that the of as a of EGFR trafficking is limited to the analysis of the early of EGFR endocytosis and that directly EGFR can be a to study receptor of endocytosis of transferrin using living cell microscopy the endosomal compartment as an of membranes (11Hopkins C.R. Gibson A. Shipman M. Miller K. Nature. 1990; 346: 335-339Crossref PubMed Scopus (316) Google Scholar). In a the fluorescent to EGFR in of this The sorting of the as EGFR, to the vesicular compartments is to be the mechanism of the lysosomal C.R. Sci. Full Text PDF PubMed Scopus Google Scholar). with EGFR-GFP in living cells demonstrate that EGFR is in the and the vesicular of the This is in with the of efficient recycling of complexes from early endosomes A. Krolenko S. Kudrjavtceva N. Lazebnik J. Teslensko L. Soderquist A.M. Nikolsky N. J. Cell Biol. 1991; 112: 55-63Crossref PubMed Scopus (91) Google Scholar). types of of organelles were observed. rapid movement of the the is This endosomal movement is and is in several M. H. Hopkins C.R. Cell PubMed Scopus Google Scholar, N. G. J. J. Cell Biol. 1994; Scopus Google Scholar). A of endosomal and the fusion of with the and vesicular compartments within the tubular-vesicular endosomes also The of endosomal membranes is with the of the biochemical that the of the endosomal fusion Ullrich M. K. I. C. M. Nature. 1996; PubMed Scopus Google Scholar). the that the movement of within the compartments and the and fusion occur at the early stages of endocytosis. The of endosomal dynamics in that the by of several demonstrate that visualization of the by and visualization of endosomal compartments. The of of GFP with properties that it may be possible to the of the interaction of the EGFR with adaptor and in in Fluorescence with EGFR-GFP and proteins are in in and for the are to Carpenter and Beguinot for the of
Carter et al. (Tue,) studied this question.