17, Issue of June 15, pp. 11083-11094,1991. Printed in U. S. A. The Role of Tyrosine Kinase Activity in Endocytosis,. Compartmentation, and Down-regulation of ...
THEJOURNAL IC) 1991 by
OF BIOLOGICAL CHEMISTRY
Vol. 266, No. 17, Issue of June 15, pp. 11083-11094,1991 Printed in U.S.A.
The American Society for Biochemistry and Molecular Biology, Inc
The Roleof Tyrosine Kinase Activity inEndocytosis, Compartmentation, andDown-regulation of the Epidermal Growth Factor Receptor* (Received for publication, January 14, 1991)
H. Steven Wiley$$, John J. Herbst$, Brenda J. Walsh$, Douglas A. Lauffenburgerv, Michael G.Rosenfeld((**$$,and Gordon N. Gill** From the $Divisionof Cell Biology and Immunology, Department of Pathology, University of Utah Medical Center, Salt Lake City, Utah 84132, the QDepartmentof Chemical Engineering, University of Illinois, Urbana, Illinois 61801, and the 11 Howard Hughes Medical Institute and **Schoolof Medicine, University of California, S u n Diego, La Jolla, California 92093
cally importantligandssuchastransferrin (Tf)’ and low density lipoprotein (LDL), the mechanisms involved in downregulation of signal transducing receptors with intrinsic tyrosine kinase activity remain an important unsolved question in cell biology. The Tf and LDL receptors, whose primary function isbulk transport of nutritional molecules into thecell (Kaplan, 1981), cluster about coated pits which constitute approximately 2% of the cell surface (Andersonet al., 1977). Internalization and trafficking of Tf and LDL receptors is independent of ligand binding because clusteringin coated pits (Beisiegel et al., 1981; Watts, 1985), receptor internalization, and subsequent recycling (Ajioka and Kaplan, 1986; Basu et al., 1981; Stein and Sussman, 1986) is observed in both the presence and absence of ligand. Because the behavior of both occupied and unoccupied receptors is similar, ligand binding does not normally altersurfacereceptornumber or theirintracellular distribution. In the caseof LDL receptors, ligand dissociates in the acidic environment of the endosomes and is targeted to lysosomes while empty receptors recycle back to the cell surface (Brownet al., 1983). In thecase of Tf receptors,ligand remains with the receptor and both recycle back to the cell surface (Klausneret al., 1983). Incontrasttonutrient receptors, signaling receptors undergo ligand-inducedconformationalchangesthatalter their endogenous enzymatic activity and distribution. Empty EGF receptors are randomly distributed at the cell surface (Willingham et al., 1983), butupon binding ligand they cluster incoatedpitsandareinternalized(Haigler et al., 1979). Ligand binding also leads to activation of receptor tyrosine kinaseactivityand adecrease in surfacereceptor number (down-regulation). Down-regulation of EGF receptors attenThe cell surface is a dynamic structure in which specific uates signal transduction (Chen et d., 1989; Wells et al., 1990), components are continually added and removed. Among the and requires receptorkinaseactivity,butthemechanistic most extensively regulated cell surface components arerecep- basis of the kinase requirement is controversial (Glenney et tors because they constitute the primary means by which cells al., 1988; Honegger et al., 1987a). We have proposed that in perceive their environment.Although significant progress has the caseof full length EGF receptor, activation of its intrinsic been made indefiningthemechanismsthatregulatethe tyrosine kinase activity leads to high affinity binding to coated internalization and sorting of receptors that bind metaboli- pits(Chen et al., 1989; Glenney et al., 1988; Lund et al., 1990b). The resulting increase in internalization rate shifts receptors to an intracellular compartmentwhich is then tar*Thesestudies were supportedinpart by NationalInstitutes of Health (NIH) Grants DK 33602 (to H. S. W.), AM 13149 (to geted to lysosomes (Stoscheck and Carpenter, 1984; Wiley,
Occupancy-induced down-regulation of cell surface epidermal growth factor (EGF) receptors attenuates signal transduction. To define mechanisms through which down-regulation of this class of growth factor receptors occurs, we have investigated the relative roles of ligand-induced internalization and recycling in this process. Occupied, kinase-active EGF receptors were internalized through a high affinity, saturable endocytic system at rates up to 10-fold faster than empty receptors. In contrast, full length EGF receptors lacking tyrosine kinase activity underwent internalization at a rate independent of occupancy. This “kinase-independent”internalization rate appeared to reflect constitutive receptor internalization since it was similar to the internalization rate of both receptors lacking a cytoplasmic domain and of antibodies bound to empty receptors. EGF internalized by either kinaseactive or kinase-inactive receptors was efficiently recycled and was found within endosomes containing recycling transferrin receptors. However, targeting of internalized receptors to lysosomes did not require receptor kinase activity. All receptors that displayed ligand-induced internalization also underwent downregulation, indicating that the proximal cause of downregulation is occupancy-inducedendocytosis. Tyrosine kinase activity greatlyenhances this process by stabilizing receptor association with the endocytic apparatus.
G. N. G.), and National Science Foundation Grant BCS89-17010 (to D. A. L.)as well as Grant CD-456Nfrom the American Cancer Society (to G. N. G.) and a grant from the Markey Charitable Trust. § Recipient of an NIH Research Career Development Award. To whom correspondence should be addressed Dept. of Pathology, University of Utah Medical Center, 50 N. Medical Dr., Salt Lake City, U T 84132. $$ An Investigator of the Howard HughesMedical Institute.
The abbreviations used are:Tf,transferrin; IS-, intracellular sequence negative; LDL, low density lipoprotein; PVP,polyvinylpyrrolidone; k,, specific endocytic rate constant; k,, recycling rate constant; kh, ligand degradation rate constant; EGF, epidermal growth factor; PBS, phosphate-bufferedsaline; HEPES, 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid HRP, horseradishperoxidase; DAB, diaminobenzidine.
11083
11084
Internalization Down-regulation and
of the EGF Receptor
1985). However, other investigatorshave reported thatligand- A modified dihydrofolate reductase gene was the selectable marker forall transfections. B82 cells were grown in Dulbecco's modified induced internalization of the EGF receptor is independent Eagle'smedium (Flow Laboratories)containing dialyzed 10% calf of tyrosine kinase activity and that down-regulation is due serum (HyClone). 5 p~ methotrexate was added to the medium for t o a kinase-mediated inhibition of EGF receptor recycling those cells transfected with human EGF receptor. (Felder et al., 1990; Honegger et al., 1987a,1990). A third Binding Studies-Cells grown to confluence in 35-mm dishes were to serum freeDulbecco's modified possibility is that receptor kinase activity is involved in both switchedfromgrowthmedium Eagle's medium containing 20 mM HEPES (pH7.4) and 0.1% bovine endocytosis and postendocytic compartmentation.Distinguishing between these alternate possibilities is important serum albumin and no bicarbonate (DHB) 18 h before experiments. Binding experiments were initiated by changing to DHB containing because little is known regarding the mechanisms of ligand- the indicated concentrationsof labeled ligand. The additionof ligand induced endocytosis or the processes that regulate postendo- and the rinsing of cells were done with a semiautomatic apparatus cytic sorting and targeting. In addition, each would require (Wiley and Cunningham, 1982). The temperature limits for binding that an entirely different strategybe taken to determine the experiments were 35.5-37 "C,and the temporalresolution was within 10 s. Binding was terminated by rapidly rinsing six times with 2 ml molecules involved in ligand-dependent down-regulation. To accurately determine the rate of ligand-induced endo- of ice-cold WHIPS buffer (20 mM HEPES (pH 7.4), 130 mM NaC1, 5 KC1,0.5 mM MgCI,, 1 mM CaC12,1mg/ml polyvinylpyrrolidone). cytosis, several factors must be considered. Unless a receptor mM The relative amounts of ligand associatedwiththesurfaceand is specifically excluded from a coated pit, it shouldalways be interior of the cells was determined by acid stripping at 0 "C using 50 subject to random entrapment (Goldsteinet al., 1984). Thus, mM glycine-HC1, 100 mM NaC1, 2 mg/ml PVP, 2 M urea (pH 3.0). simply observing a finite endocytic rate for a hormone recep- For pulse-chase studies in which viability of the cells after stripping tor does not prove that the process is ligand-induced. In was important, the stripping solution omitted the urea. Stripping addition,receptor-mediated endocytosis is asecond order efficiencies were determined in parallel and were generally 98% for theureasolutionsand 90% inthe absence of urea. Nonspecific process in which receptors compete for binding to componentsbinding was determined in the presence of at least 200-fold molar of coated pits or other endocytic structures (Pearse, 1988; excess unlabeled ligand in the case of Tf, or by measuring binding to Wiley, 1988; Lund et al., 1990b). Thus the extentof receptor B82 cells that lack EGF receptors in thecase of labeled EGF and528 occupancy influences the fraction of occupied receptors en- monoclonal antibody. Nonspecific binding wasgenerallyless than tering thecoated pit pathway.Once the endocytic components 5% of total binding. Single label experiments using "'I-labeled 528 IgG in the absence and presence of EGF established that EGF did aresaturated, receptor internalizationcan onlyproceed affect the specific internalizationrate of theantibody. Cell through a nonspecific mechanism. The effect of ligand recy- not number was determined with a Coulter Counter. cling and degradation mustalso be considered. If these procFluid Uptake Studies-Cells were treated as described above for esses are fastrelative to endocytosis, then estimatesof ligand binding studies, except that ["'I]PVP was used. Cells were changed internalization rates will be too low (Wiley and Cunningham, a t 37 "C to the indicated concentrationof ["'I]PVP and at appropriate times the medium was aspirated and the cells rinsed 10 times at 1982; Waters et al., 1990). 0 "C withWHIPS solution. Cells were then removed from their plates In the present study, we have examined the relationship trypsinizationand collected by centrifugation(McKinleyand between ligand-induced internalization, recycling, and down- by Wiley, 1988). The amount of fluid incorporation was corrected for regulation of the EGFreceptor. We have also determined the cell number. influence of receptor tyrosine kinase activity on these procDown-regulation-To evaluate the ability of 528 monoclonal IgG esses. Using labeled monoclonal antibodies to follow unoccu- to down-regulate surface receptor expression, cells were incubated pied receptors, we found that empty receptorsundergo endo- with 13 nM 528 IgG in standard binding medium a t 37 "C. At approcytosis at the same rate as receptorslacking the cytoplasmic priate times, cells were removed from the plates by scraping and by trituration. After centrifugation, aliquots of approxidomain, indicative of random entrapment.Occupancy of EGF dispersed mately IOGcells were resuspended in 50 p1 of 528 monoclonal IgG at receptors lacking kinase activity did not change their intera concentration of 80 pg/ml. After 30 min on ice, cells were washed nalization rate. However, occupancy of kinase-active recep- twice with PBS and then fluorescein isothiocyanate-conjugated antitors increased their internalization rate up to 10-fold. Both mouse IgG (50 p1 of 20 pg/ml) was added for another 30-min incutypes of receptor recycled back to the cell surface a t similar bationon ice followed by washing with PBSand fixing in 1% rates. Truncated, kinase-inactive receptors could undergo ac- glutaraldehyde in PBS. 5000 cells per sample were analyzed using a fluorescence-activated cell sorter with FACScan celerated degradationwhen internalized due to ligand-induced Becton-Dickinson software. Mean fluorescence channels as the measurement of fluoresendocytosis, indicating that tyrosine kinase activity is not cence intensity were converted to a linear scale between 1 and lo4 required for lysosomal targeting. We conclude that ligand- and reported as relative fluorescence units. Background fluorescence (receptor negative cells stained as described above) was subtracted induced EGF receptor down-regulation is mediated through from the relative fluorescence units. alterations in endocytic rather than recycling rates. EXPERIMENTALPROCEDURES
General-Mouse EGF was purified from submaxillaryglands (Savage and Cohen, 1972). EGF, human, iron-loaded diferric Tf (Calbiochem-BehringCorp.),and monoclonal antihumanEGFreceptor antibody 528 IgG (Gill et al., 1984) were iodinated with either la11 or '"'1 (Amersham Corp.)usingIODO-BEADS (Pierce ChemicalCo.) according to the manufacturer's recommendations and freeiodine separated from the radiolabeled ligands by dialysis or by passing the mixture over a 0.8 X 20-cm column of Sephadex G-10 equilibrated with PBS. The specific activity of "'I-labeled EGF was generally between 600 and 1800 cpm/fmol, "51-labeled Tf was between 780 and 3100 cpm/fmol, and '2511-labeled528 monoclonal was between 1300 and 1900 cpm/fmol. The ""I-EGF was between 240 and 430 cpm/ fmol. The fluid phase marker, ["511]polyvinylpyrrolidone ([""IIPVP) was synthesized as previously described (McKinley andWiley, 1988). Cell Culture"B82 mouse L cells, which contain noendogenous EGF receptors, and B82 cells transfectedwithnormal (WT) or mutated (MY2', c'647, c'973, ~'1022, and M"'c'1022) human EGF receptors were generated as previously described (Chen et al., 1989).
EGF-induced down-regulation was also evaluated by incubating cells either with or without 50 nM "'I-EGF for the times indicated in the figure legends. Cells were then rapidly rinsed five times with icecold WHIPS solution and brought to equilibrium with 50 nM12'1EGF (3-9 h). Cells were again rinsed and the amount of surfaceassociated ligand determined by acid stripping. Degradation of EGF-Cells were incubated a t 37 "C with 0.5 nM "'I-EGF in DHB binding mediumusingabovine serum albumin concentration of 100 pg/ml. At appropriate intervals, 100-p1 samples were collected from the medium and loaded on 12.5% native tube polyacrylamide gels running at 4 "C (Wiley and Cunningham, 1982). Free iodine, iodotyrosine, and EGFwere cleanly separated in thegels by isotactophoresis. Gels were sliced and the amount of degraded EGF quantified in the iodotyrosine peak. No free iodinewas generated during incubation of cells with EGF. In addition, no degradation was observedwhen EGF was incubated with parental B82 cells which lack endogenous EGF receptors. Gradient Fractionation of Cells-Subconfluent monolayers of cells grown in 100-mm plates were incubated with radiolabeled ligand at the indicated times a t 37 "C. After rinsing, surface-associated ligand was removed with 100 mM NaC1, 50 mM glycine (pH 3.0) for 2 min
Internalization Down-regulation and
of the EGF Receptor
11085
followed by rinsing with saline and100 PM phenylarsine oxide for 10 approach, an EGFreceptor mutant that lacked a cytoplasmic min at 0 "C to render thecells fragile. Cells were removed from their domain was constructed because information that specifies plates by incubationwith 600 pg/ml trypsin a t 0 "C followed by high efficiency internalization for other receptors is located neutralization with5-fold excess soybeantrypsin inhibitor.Cells were pelleted and homogenized in 1 ml of 250 mM sucrose, 10 mM trieth- in this region (Davis et al., 1987; Jing et al., 1990; Verrey et al., 1990). The internalization rate of this "intracellular-seanolamine, 10 mM acetic acid, 1mM EDTA (pH7.4), by eight passages through a 25-gauge needle. Cell breakage was generally 80-90% as quence-minus" (IS-) receptor should reflect random receptor assayed by microscopy. Nuclei were removed by centrifugation a t 900 entrapment by either smooth or coated pits. In the second X g for 3 min. Samples were loaded on linear 19-40% (w/w) sucrose/ approach, a monoclonal antibody (mAb 528) was used as a PVP gradients and brought to isopynic equilibrium by centrifugation marker to follow empty receptors. This antibody does not in an SW 40 rotor at 37,500 rpm for 12 h a t 4 "C (Opresko et al., activate the receptor and cannot bind to occupied receptors 1980). Gradients were pumped out of the top of the tubes using an (Gill et al., 1984). This allowed use of a double-label protocol ISCO model 185 density gradient fractionator a t a rate of 1 ml/min. Fractions of 0.3 ml were either counted or evaluated for enzymatic tosimultaneously observe both occupied and unoccupied activity. Samplescontainingboth '"I and ""1 were countedon a receptors. three-channel Packard gamma spectrophotometer. All counts were Wepreparedthe IS- EGFreceptortruncationmutant corrected for channel spillover and counting efficiency. Sedimenta(c'647) which retains only a two-amino acid C' terminus past tion position of the plasma membranewas determined by binding of '"'I-labeled wheat germ agglutinin orby surface iodinationusing sulfo- the transmembrane domain and evaluated the abilityof this As S H P P (Pierce Chemical Co.) a t 0 "C. Both methods yielded ident,ical receptor to both down-regulate and to internalize EGF. results. Endosomes were identified by incubating cells to a steady shown in Fig. 1, the IS- EGF receptor was internalized at a state with'"I-Tf followed by removal of the surface-associatedligand low, but significant rate. EGF did not induce down-regulation with glycine-HC1 prior to cell fractionation. Lysosomes were identi- of this mutant receptor. We also examined the kinetics of fied by hexosaminidase activity (Horvatet al., 1969) and mitochondria both internalization and down-regulation of full length kiby cytochrome c oxidase activity(Sottocasa et al., 1967).Density nase-activeandkinase-inactiveEGFreceptorsunderthe profiles were determined by refractive index. Density Shiftingof Endosomes-Colocalization of different ligands withinendosomes was established by thehorseradish peroxidase Down-regulationInternalization (HRP)-diaminobenzidine (DAB) density shift technique (Courtoyet al., 1984; Ajioka and Kaplan, 1987). The conjugate between Tf and HRP (HRP.Tf) was a gift of Dr. Jerry Kaplan. A 1:l conjugate of EGF and HRP (HRP. EGF) was prepared by the method of Nakane and Kawaoi (1974). The HRP was purified by CM-cellulose chromatography (Shannon et al., 1966) followed by conjugation to EGF. The HRP.EGFwas isolated by sequential chromatography on P-100 (Bio-Rad) and CM-cellulose using a 0.05-0.1 M sodium acetate gradient (pH 4.4). The peak eluting at 0.06 M acetate was confirmed to be a 1:l EGF.HRP conjugateby absorbance ratio at 280 and 400 nM as well as enzymatic activity and ability to compete with "'1-EGF for receptor binding. Cells in 100-mm dishes were incubated with 50 nM nM of either "'I-EGF or l2.'Iof either HRP. Tf or HRP. EGF and 50 Tf for 60 min a t 0 "C followed by a shift to37 "C for 15 min. Surface- 100 f "21 (Kin -) associated ligand was removed by acid stripping and the cells were 2. e. homogenized using 10 passages through a ball-bearing homogenizer -EO (BalchandRothman,1985).Thepostnuclearsupernatants were c a treated with DAB and H20, (Ajioka and Kaplan, 1987) and then -60 fractionated by isopynic sucrose gradients as described above. Receptor Degradation-Cells expressing the indicated mutant EGF 0 receptors were treated with or without50 nM EGF for 24 h at 37 "C. -40 0 Duplicate plates of cells were harvested using hot Laemmli sample ' 2 buffer, pooled and resolved on 7.5% sodium dodecyl sulfate-polyacryl20 amide gels, transferred toImmobilon membranes, and EGF receptors were detected using either monoclonal antibody C13 or polyclonal antibody N-20 directed against peptides corresponding to residues 996-1022 and 1-20 in the hEGF receptor, respectively. Antibodies were diluted 1:10,000 and visualized with an alkaline phosphatasecoupled second antibody. Pulse-chase experimentsusing cells labeled with ['"SS]methionine were performed asdescribed (Chen et al., 1989). Data Analysis-Values for surface bound and internalized ligand were corrected for nonspecific binding and forspillover from the interior and surfaceof the cell, respectively (Wiley and Cunningham, 1982). Specific internalization rates were determined by plotting the integral of surface-associated ligand against the amountint.ernalized 0 25 75 50 100 0 30 60 90 (Opreskoand Wiley, 1987). The slopes were thendetermined by JSurface (01.ofTime total) (min) linear regression. Correlation coefficients of internalization plotswere generally >0.98. Statistical analysesof the datawere performed using FIG. 1. Internalization and down-regulation of EGF recepa standard t test, estimating the varianceof each set of data individ- tors. Cells expressed the c'647 IS- truncation ( t o p ) , M7" kinaseually. Confidence intervals for each comparison aregiven in the text. inactive (middle),and wild type kinase-active (lower)EGF receptors. All statistics were calculated using the Data Desk program (Odesta Leftpanels, cells were incubated simultaneously with0.8 nM ""I-EGF Corporation). Satin plot analyses were performed as previously de- ( 0 )and 1 nM '""I-labeled 528 monoclonal antibody (0).The specific scribed (Lund etal., 1990b). Data were fitto a two-component internalization rate of the ligand was determined over a 5-min time internalization system using the Simplex algorithm. period. All data are on the samescale and are presented as internalization plots. Right panels, cells were treated with either 10 nM 528 monoclonal antibody (0)or 16 nM "'I-EGF (0)for the indicated RESULTS times. Cells were then rapidly cooled and brought toequilibrium with The Constitutive Internalization Rate of the EGF Receptortheir respective ligand. The number of surface receptors was deterT o determine the basal rateat which unoccupied EGF recep- mined by either flow cytometry for the monoclonal antibodyor tors are internalized, two approaches were used. In the first surface strippingfollowed by counting for EGF.
E
1
Y
"
11086
of the EGF Receptor
Internalization Down-regulation and
same conditionsused to evaluate the IS- construction. Kinase activity was required for both efficient internalization and down-regulation (Fig. 1).The average internalization rate of the kinase-active receptor (0.32 min") was 8-fold higher than the M7" kinase-inactive receptor. Significantly, the internalization rate of EGFboundto full lengthkinase-inactive receptors was indistinguishable from that observed with the IS- receptor. The specific internalizationrates of anti-EGFreceptor antibody bound to wild-type, kinase-inactive, and IS- receptors were all similar (Fig. 1).These rates (0.03-0.05min") were also similar to those displayed by EGF bound to both kinase-inactiveand IS- EGF receptors. Concentrations of antibody ranging from 0.1 to 12 nM did not affect internalization rates incells expressing wild-type EGF receptors(0.03 min"). In addition, these concentrationsof antibody did not induce receptor down-regulation (Fig. l ) , in agreement with previous studies using this antibody (Kawamotoet al., 1983). A statistical summary of receptor internalization rates at low concentrations of EGF and anti-EGF receptor antibody is presented inFig. 2. These data were collected over a period of 2 years and show the total rangeof observed values. There was no significant difference ( p > 0.05) between the internalization rate of EGF bound to either the full length kinaseinactive or IS-receptors. In addition, therewas no difference between the rate of antibodyinternalization displayed by kinase-active, kinase-inactive, or IS- EGFreceptors. The only significant differencewasbetween the internalization rate displayed by kinase-activereceptorsbindingEGFandall other receptors ( p < 0.01). Induced Internalization of the EGF Receptor Is SaturableT o avoid saturation of the specific, coated pit pathway(Wiley, 1988), the internalization datashown in Fig. 2 were collected using 0.2 nM EGF. At this concentration, the internalization rate of wild type EGF receptors was 8-10-fold higher than that of kinase-inactive receptors. Increasing concentrations of EGFresultedin aprogressivedecrease in the specific internalization rate of the wild type receptor (Fig. 3A). These data are presented asa Satin plot, where the slope is proportional to the affinityof receptors for the endocytic apparatus
.-
0.40
0.40
B
1
0.30
z. $ Dl
0.20
s. 0
2 0.10 3,
=,
0.0
6.0
12
18
0.0
6.0
12
18
:
0.00
Internalizationvelocity (rnolecules/cell/rninXIO")
FIG. 3. Tyrosine kinase activity is required for utilization of the high affinity endocytic system. A , specific and net internalization rateswere determined for the c'647 truncation (A),kinaseinactive (O), and wild type ( 0 )EGF receptors using ligand concentration ranging from0.17 to 18 nM. Included is internalization of labeled 528 monoclonal IgG bound to wild type receptors (+) using concentrations rangingfrom 0.13 to 13 nM. The data are plotted asa Satin plotwhich is analogous to a Scatchard plot. The curve through the data was fit to a two-componentinternalization model (high affinity and constitutive internalization) using the Simplex algorithm. B , Satin plot analysis of kinase-active (m) and kinase-inactive (0) c'1022 truncation mutations.
(Lund etal., 1990b). TheSatinplot of thekinase-active receptor was biphasic, indicating entry through both a high affinity and constitutive endocytic pathway (Heisermann et aL, 1990; Lund et al., 1990b). The affinity of the high affinity component was 2.5 X per receptor. Satin plots of the ISandkinase-inactivereceptors were virtuallyidenticaland displayed a slope of zero (Fig. 3A), as would be expected of receptors unable specifically to bind to coated pit components. The slope of the Satin plot of wild-type receptors using IZ5Ilabeled 528 IgG as a ligand was also zero (Fig. 3A), indicating that binding of the antibody to unoccupied EGF receptors does not affect their interaction with the endocytic apparatus. The data in Fig. 3A indicate that the protein tyrosine kinase activity of the EGF receptor is necessary for specific receptor binding t o endocytic structures. However, we previously described akinase-inactive truncation mutantof the EGFreceptor (M721~'1022) that is ableundergo to ligand-induced internalization anddown-regulation (Chen et al.,1989; Lund et al., 1990a). T o determine whether the M7*lcr1022 mutant was able to undergo ligand-induced binding to the endocytic apparatus, Satin plot analysis was performed using both this mutant and its kinase-active parent receptor (~'1022).As shown in Fig. 3B, the M721~'1022receptor displayed a low, but significant slope ( p < 0.01). In multiple experiments, endocytic affinities were consistently observed to fall between 0.3 .per receptor, indicating that the M7%'1022 4.3 and 7.7 X a mutant EGF receptor has a low affinity for the endocytic apparatus relative to wild type receptors. However, this low affinity was significantly greater than that displayed by ISor holo kinase-inactive receptors (Fig. 3A). As expected, the kinase-active ~ ' 1 0 2 2receptor displayed a higher affinity for endocytic structures than the kinase-inactive c'1022 mutant 0.0 (Fig. 3B). WT M7" c'647 WT M721 c'647 Satin plots will yield a negative slope for a given receptor EGF 528 IgG type when occupancy increases the internalization rateof the FIG. 2. Statistical summary of the internalization rates of receptor (Lund et al., 1990b). All EGF receptors thatdisplay both occupied and empty EGF receptors. All specific internali- endocytic saturation should thus internalize EGF faster than zation rates of the different receptors were determined using either simultaneously bound 528 antibodies. As shown in Table I, 0.2 nM EGF or 1 nM 528 monoclonal IgG for the indicated cell types. this was observed. Antibodybound to kinase-active or kinaseThe data are presented aasbox plot in which the center is the median inactive c'1022 EGF receptorswas internalized slightly faster value, while the box encloses the middle 50% of the values, the shaded region is the standard deviation, and the wiskers enclose all the data than antibody bound tofull length receptors. However, internalization of EGF bound to either c'1022 receptor was signifvalues. Values in parentheses are the total number of internalization icantly faster than antibody internalization ( p < 0.01). Alplots from separate experiments included in the analysis.
Internalization Down-regulation and
of the EGF Receptor
TABLEI Saturation of specific endocytosis is associated with induced internalization Cells were incubated for 5 min a t 37 "C with a combination of '"'1EGF and 'YsII-labeledanti-EGF receptor monoclonal antibody 528. The labeled EGF was at the indicated concentration, while the 528 monoclonal antibody was at a concentration of 1.3 nM. Samples were taken at 1-min intervals. The specific internalization rates ( k c , )of the indicated ligands were determined from internalization d o t s . Receptor concentration type
'"I-EGF
"21
0 0.8 17
by
'\ 0
\ O'..
k,
EGF
1'511-528 IgG
min"
nM
WT
11087
0.26 0.09
0.05 0.06 0.05 0.04 0.04
0 0.8 0.04 17
0.03 0.02
c'1022
0.8 17
0.27 0.14
0.08 0.07
M7"c'1022
0.8 17
0.13 0.10
0.07 0.07
0-l 0
I
5
10
15
Time (rnin)
FIG. 4. Fluid phase endocytosis and diacytosis in B82 cells. Confluent cultures of cells were incubated with ['''I]PVP at a concentration of 4.5 X 10"cpm/ml for the indicated times and the amount of internalized solute was determined after removing the cells from their plates (0).The labeled medium was also removed and replaced with unlabeledmedium a t 5 and 10 min after which the amount remaining inside thecells was determined ( 0 ) The . inset is the chase data plotted on a semilog scale.
though high concentrations of EGF could reduce the specific depend on incubation time, but the fraction of fluid phase internalization rate of receptors occupied with EGF, there marker that was retained by the cells increased with the was no effect on the internalization rate of receptors complexed with 528 antibody (Table I). These data are consistent incubation period. These results are consistent with previous with previous observations that internalization of EGF recep- studies on fluid phase diacytosis and demonstrate that B82 cells are capable of rapid membrane recycling on the time tors is anoncooperativeprocess(Wiley and Cunningham, 1982; Waters etal., 1990) and thatoccupied receptors compete scale of the experiments. They also set an upper value of K, with each other for internalization (Lund etal., 1990b; Wiley, of approximately 0.19 rnin". To determine whether receptor-associated ligands were rap1988). which previously interRecycling and Degradation of EGF AreIndependent of idly recycled, we examined the rate at Receptor Tyrosine Kinase Actiuity-The internalization plot nalized Tf and EGF were returned to the medium. Since Tf rapidly recycles while attached to its receptor, its use as an method for determining receptor internalizationratesassumes that there is no loss of ligand from within the cell internal control permitted direct comparison between cells during the time of measurement (Opresko and Wiley, 1987), expressing kinase-active and kinase-inactive EGF receptors. a n assumption that has been validated in other cell types EGF was labeled with ""1 and Tf was labeled with '"I. Cells (Dunn and Hubbard, 1984; Wiley and Cunningham, 1982). expressing either kinase-active or kinase-inactive receptors T o ensure that differences between the behavior of kinase- were exposed to a mixture of both ligands for 2 min followed active and -inactive receptors were not due to differences in by a chase in medium containing ahigh concentration of their rateof recycling or ligand degradation, these parameters unlabeled ligands to preventradiolabeled ligand rebinding.As shown in Fig. 5B, the recycling and discharge of Tf was were directly measured. As demonstrated under"Appendix," the difference between identical for cells expressing either kinase-active or kinaseof ligand return in this experthe "true" specific internalization rate ( k , )and the"observed" inactive EGFreceptors. The t8,2 value provided by internalization plots [ ( kr)ohsJis a function iment was approximately 5 min, which was slower than the of k, and kh, where k, is the rate constantof recycling and kh rate of fluid phase diacytosis. In multiple experiments, the measured time scale of Tf recycling was between 5 and 12 is the rate constantof ligand degradation: min in B82 cells, which is typical for this receptor (Jing etal., (k,),,", - ( k , + k h ) t (1) 1990; McGraw and Maxfield, 1990). (kp)Ohs 1 - e"kr+kh" EGF was also rapidly lost from the cells, but at a slower rate than Tf. The loss of Tf could be detected within 6 min If these parameters are sufficiently small, then measurements of kc,will be accurate to within a known degree. How- of the initial ligand exposure, while loss of EGF was detected only after 8 min. In the experiment shown in Fig. 5, EGF ever, by measuring ligand recycling and degradation rates, internalized by cells expressing kinase-active receptors was one can correct the"observed" internalization rate. lost witha t,/!of 12 min, while cells expressing kinase-inactive To determineanupperlimit for the recycling ratein of 25 min. The average of five transfected B82 cells, we measured the rate and extent of receptors lost EGF with a of EGF loss of 14 f 2 and fluid phase diacytosis. Diacytosis, the return of nonspecifi- separate experiments showed a t8,A cally incorporated solute back to the medium, provides an 16 5 7 min for full length kinase-active and -inactive recepupper limit to the rate which at cells are capable of recycling tors, respectively. These differences were notstatistically significant ( p > 0.05). The average recycling rate of EGF (Besterman et al., 1981; McKinley and Wiley,1988).Cells internalized by either kinase-active or kinase-inactive c'1022 were incubated for 5 and 10min with ['"'IJPVP and then chased inunlabeled medium. The amountof material remain- truncation mutations was 10min (data not shown). Thus, ing with the cells was then measured. As shown in Fig. 4, the although structural featuresof the EGFreceptor could play a cells rapidly returned the ['2sIJPVPback to the medium with role in regulating the rate of recycling, intrinsic kinase activity an apparent b,- of3.6 min. The rate of diacytosis did not does not appear tobe a primary regulator of this process. "
b
2
Internalization Down-regulation and
11088
of the EGF Receptor Bottom H
PM
m 0
-2
2
H
Mit
Lys
Transferrin
[Pulse
2
0
-1.11
-
1
B \
Transferrin
40
2o Ipulse y\fl
10
0
6
12
,.
18
Time (min)
FIG. 5. Recycling of EGF and transferrinby B82 cells. Cells expressing either the wild type (0,B) or kinase-inactive EGF receptors (0,O) were incubated with a combination of ""I-EGF ( 0 , O )and ""I-Tf (W, 0 ) for 2 min and then chased for the indicated times in the presence of excess unlabeled ligand to preventradiolabeled ligand rebinding. The amount of ligand remaining inside the cells was then determined by acid stripping.
0
10
20
30
40
Fraction No.
FIG. 6. Colocalization of EGF and transferrin in recycling Intracellular Compartmentation of EGF Relative to Trans- endosomes. A , sedimentation of endosomal transferrin. Cells were ferrin-Although recycling of EGF through thecell was qual- incubated for either 5 (solid line) or 25 min (dotted line) with 6.5 nM itatively similar to that of Tf, itwas unclear whether thetwo '""ITf. Surface ligand was removed and cells were fractionated on ligands were using the same intracellular route. To clarify this 15-50% isopycnicsucrose gradients.Parallel groups of cells were issue, cells were incubated for either 5 or 25 minwith a incubated a t 0 "C with "'I-labeled wheat germ agglutinin tolabel the membrane ( E " ) . The majority of mitochondrial and lysosocombination of '"I-Tf and 13'1-EGF. Surface-associated li- plasma mal enzyme activity sedimentedto the bottomof the gradient except gand was removed at 0 "C followed by cell homogenization for minor peaks designated M i t and Lys, respectively. B , sedimentaand fractionation. The distribution of EGF and Tf across the tion of endosomal EGF. Cells were incubated for either 5 (solid line) endosomal region of the density gradients was then evaluated. or 25 min (dotted line) simultaneously with 2 nM ""I-EGF and 6.5 As shown in Fig. 6, endosomes were distributed over a rela- nM "'I-Tf. Surface ligand was removed and endosomeswere fractiontively symmetrical peak centered at a density between 1.12 ated as in A. Shown is the distribution of internalized l:"I-EGF. The ratio of EGF/Tf was constant across the gradient. C, same experiment and 1.14 g/ml, while the plasma membrane was found in the as in A except cells expressed kinase-inactive receptors and the I I I I more dense region of thegradient (1.15-1.16 g/ml).The EGF concentration was at 20 nM. The ratioof EGF/Tf was constant lysosomal marker hexoseaminidase was well separated from across the gradient. the endosomes and distributed asa bimodal peak at 1.16 and 1.21 g/ml. Internalized EGF cosedimented with the endoso- incubated with either HRP. Tfor '"I-Tf and thenmixed prior mal Tf after either a 5-min or 25-min incubation, indicating to homogenization, no density shift occurred (Fig. 7B). In a that the samerecycling pathway was utilized by both ligands. similar manner, endosomes containing both HRP.EGF and There was no difference in the compartmentationof EGF in 12'I-EGF could be shifted to the denseregion of the gradient cells expressing either kinase-active or kinase-inactive EGF by DAB-H202 treatment (data not shown). These data conreceptors (Fig. 6), indicating that postendocytic compartmenfirm that density shiftingoccurs only when ligand and HRP tation is independent of intrinsic receptor tyrosine kinase are located in the samevesicle (Ajioka and Kaplan, 1987). activity.Although the average density of EGF-containing To demonstrate the colocalization of Tf and EGF within endosomes shifted to a lower density after 25 min, the same endosomes,cells expressing either kinase-active or kinasewas observed for internalized transferrin (Fig. 6). To determine whether both Tf and EGF were in the same inactive EGF receptors were incubated simultaneously with endocytic vesicles, we utilized thehorseradish peroxidase 12'I-EGF and HRP.Tf for 60 min at 0 "C followed by a chase DAB density shift technique (Courtoyet al., 1984; Ajioka and for 15 min at 37 "C. As shown in Fig. 7, C and D , the HRP. Kaplan, 1987). This method is based on peroxidase-H2O2- Tf was able to shift allendosomally localized "'I-EGF to the catalyzed oxidation of DAB within vesicles. The dense poly- bottom of the gradient. Significantly, there was no difference by either mer of DAB which forms within the vesicle lumen increases in the abilityof HRP.Tf to shift EGF internalized the buoyant densityof the vesicle. Thus, peroxidase-contain- the kinase-activeor kinase-inactive EGFreceptor. These data ing vesicles can be separated from other vesicles by density indicate that the EGF-receptor complex traverses the same recycling endosomal pathwayin B82 cells as does the Tf gradient centrifugation. Conjugates of HRP and both EGF receptor. In addition, the protein tyrosine kinase activity of and Tf were used to place peroxidase activity within endosomes containing the respective receptors. As shown in Fig. the EGF receptordoes not appear to qualitatively dictate its 7A, incubation of cells simultaneously with bothHRP. Tf and postendocytic compartmentation. l25II-Tfresulted in a density shift of endosomal '"I-Tf after Relative Extentof EGF Degradation versusRecycling-Loss DAB. H2O2 treatment. This density shiftrequired the pres- of previously internalized EGF by the cell could be due to ence of HRP. Tf (Fig. 7 B ) .When separate platesof cells were either ligand recycling, rapid degradation, or both. To deter-
Internalization and Down-regulation of the EGF Receptor HRP-TI + 1251+Tl
HRP-TI + '=I-EGF
bomm top
11089 bottom
4.0
3.0 2.0
-
-P 1.0 51
0.0 6.0 4.0
$ 5
-E
2.0 0.0 10
20
30
40
10
20
30
40
Fraction No.
FIG. 7. HRP-transferrin conjugates can density shift internalized EGF in B82 cells. Cells were incubated with 50 nMof HRP.Tf and either '""ITf (A and B ) or "'I-EGF (C and D ) for 60 min at 0 "C followed by 15 min a t 37 "C. Surface-associated ligand was removed prior to homogenization. A, density shift of ""I-Tf. Homogenates were treated either without (solidlines) or with (0)H,02 and DAB priorto sucrose gradient fractionation. The shifted material was found in the 60% sucrose cushion at the bottom of the gradient. The nonshiftahle material at the top of the gradient is ligand released during cell homogenization. B , density shift requires colocalization of HRP and ligand. Cells were either treated with '"I-Tf alone (shaded lines) or plates of cells were incubated separately withI2'II-Tfand HRP.Tf prior to mixing andhomogenization (solid lines)and then treated withH202and DAB prior to sucrose gradient fractionation. C, HRP.Tf will shift '""IEGF. Cells expressing kinase-inactive M7" EGF receptors were incubated with both l""IEGF and HRP.Tf. Homogenates were treated either without (solid lines) or with (0) H202 andDAB prior to sucrose gradient fractionation. D , same as C, hut cells were expressing wild type EGF receptors.
mine the relative contribution of ligand degradation to this process, we quantified the appearance of EGF degradation products in the medium by native gel electrophoresis. This technique is capable of detecting 0.5%of the total EGF as degradation products (Wiley and Cunningham, 1982; Wiley et al., 1985). As shown in Fig. 8A, there was no measurable EGF degradation by cells expressing kinase-active receptors until 30 min after initial ligand exposure. Cells expressing kinase-inactive receptors displayed significant EGF degradation only after 60 min. The amount of intracellular ligand reached a pseudo steady state prior to significant degradation in either cell type (Fig. 8, B and C), indicating thatat steady state, loss of internalized EGF from these cells occurs primarily by recycling and not by lysosomal degradation. If the amount of internalized EGFin B82 cells is primarily regulated by recycling, then total inhibition of degradation should haverelatively little effect on the net intracellular accumulation of ligand. This was tested by treating cells with monensin. It has been previously demonstrated that monensin will inhibit intracellular degradation of both EGF (King, 1984; Wiley et al., 1985) anditsreceptor(Stoscheckand Carpenter, 1984).Although monensin will also block the recycling of receptors inwhich ligand dissociation is a requirement for sorting ( i e . the LDL receptor (Basu et al., 1981)), it 0 30 60 90 120 Incubation time (min) has little effect on the recycling of membrane lipid (Koval andPagano, 1989) and arelatively minor effect onother FIG. 8. Accumulation and degradation of internalized EGF. or et al., 1986) or Tf A, cells expressing either kinase-inactive M"' EGF receptors (0) receptor types (i.e. insulin (Huecksteadt ( 0 )or presence of 15 receptors (Stein and Sussman, 1986)).Fig. 8A shows that 15 wild type kinase-active receptors in the absence PM monensin (A)were incubated with 1.7 nM I2'I-EGF at 37 "C. At P M monensin completely blocks EGF degradation in transthe indicated times the medium was sampled and analyzed for [""I] fected B82 cells. However, monensin had littleeffect on either monoiodotyrosine (MIT) by native gel electrophoresis. B , cells exreceptor down-regulation or internalization incells expressing pressing wild type receptors were incubated with 1.7 nM '""I-EGF in either kinase-active or kinase-inactive EGF receptors (Table the absence ( 0 )or presence of 15 PM monensin (A).At the indicated timestheamount of internalized ligand was determined by acid 11). In addition, we found that monensin had no significant stripping. C, cells expressing kinase-inactivereceptors were incubated effect on the recycling of internalized EGF, with a tjJ2of 16 with 17nM "'I-EGF in the absence(0) or presenceof 15 PM monensin min after monensin treatment. As expected, blocking EGF (A). At the indicated times the amount of internalized ligand was degradation did not cause an appreciable net accumulation of determined by acid stripping. intracellular ligand (Fig. 8, B and C). These data confirm that the majority of EGF internalized by transfected B82 cells
Internalization and Down-regulation of the EGF Receptor
11090
TABLE I1 Effrcts of monensin on EGF receptor internalization and down-regulation Cells were treated with monensin for 30 min and then for 2 h in the presence of monensin and either 1.7 nM unlabeled EGF (for the I:, experiments) or 20 nM EGF (for down-regulation). Receptor internalization rates were then determined using the same concentration of ”‘I-EGF. Percentage of surface receptors remaining after the 2-h treatment was determined by equilibrium binding of “‘I-EGF a t 0 “C using untreated cells as the 100% base line. Receptor t w
WT (Kin’)
M:” (Kin-)
Monensin
k,.
Surface receptors remaining
PM
rnin”
%
0 15 0
0.20
39
0.17
50
0.04 0.03
93 100
15
follows a recycling rather than a degradative pathway. To estimate the relative extent of recycling uersus degradation of EGF in B82 cells, we simultaneously measured the rates of EGF degradation and internalization aswell as surface-associated ligand a t steady state (time, >90 min). Degradation was determined by native gel electrophoresis and internalization was determined after thecells reached steady state with unlabeled EGF. For these experiments, cells expressing wild type receptorswere incubated with0.5 nM EGF. At steady state the cells had 13,000 molecules bound to the surface. Their specific internalization rate was 0.27 min” and 690 molecules were degraded per minute, as measured by iodotyrosine release into themedium. This translates to a net internalization rate of 3500 molecules per min a t steady state during which time less than 700 molecules were degraded. Very similar resultswere obtained for cellsexpressing kinaseinactive receptors. Thus for each five molecules of EGF internalized at steady state, oneis degraded and four are recycled. From the above data, the influence of ligand recycling and degradation on the measured specific internalization rate ( k , ) can be determined. Although no ligand recycling or degradation could be detected during the initial 5 min after ligand addition, a maximum possible value forboth k, and kh can be set by assuming no delays in recycling and degradation. This yields upper bounds of k, = 0.06 rnin”; kh = 0.01 min” for the kinase-active receptor and k, = 0.03 rnin”; kt, = 0.01 min” forkinase-inactive receptors.Because internalization was measured over a 5-min period for all cells, these values inserted in Equation 1 yield a ( kc)tn,e/(ke)“bSratio of 1.15 for the kinase-active EGF receptor and 1.09 for the kinase-inactive EGF receptor. Conservatively,the specific internalization rate of the kinase-inactive EGF receptor is thus underestimated by 9% and that of the kinase-active EGF receptor is underestimated by 15%. Induced Internalization Is Sufficient to Accelerate Receptor Degradation-It has been postulatedthattyrosinekinase activity of the EGF receptorleads to down-regulation due to phosphorylation of a protein inmultivesicular bodies required for lysosomal targeting (Felder et al., 1990). If this is the case, then ligand-induced loss of receptor mass should be dependent on receptor kinase activity, but independentof induced internalization. T o critically test this hypothesis,we examined the effect of occupancy on degradation ratesof kinase-active EGF receptors truncated toresidue 973 (c’973) and kinase-inactive M7;”c’1022receptors. The c‘973 receptors retain full tyrosine kinaseactivityand signal biological responses, but lacka specific domainrequiredforligand-induced internalization (Chen et al., 1989). AlthoughM’”c’1022 receptors lack kinase
activity, they do retain a degree of occupancy-induced internalization (Table I). As shown in Fig. 9A, EGF reduced the cellular mass of M7”’c’1022 receptors to amodest extent, concordant with the relative extent of ligand-induced internalization of this receptor (Fig. 3B). This reduction was due to an accelerated degradation rate of the M:”’c’1022 receptor as was the casewith the holo, kinase-activereceptor(Fig. 9B). In contrast, thecellular mass of c’973 receptors was not affected by EGF addition despite their high levels of intrinsic tyrosine kinase activity (Fig. 9A). These data demonstrate that kinase activity is not required for occupancy-induced receptor degradation and thus phosphorylation of a protein in multivesicular bodies cannot be a requirement for lysosomal targeting. However, kinase activity leads to an increased loss in receptor mass in parallel with effectson internalization rates. This indicates that receptor degradation is proportional to thepool of intracellular receptorswhich in turn is regulated primarily by receptor internalization. DISCUSSION
A basic question regarding the diverse classes of membrane receptors thatpossess intrinsic tyrosinekinase activity is the molecular mechanisms by which they are down-regulated. While it has been firmlyestablished that the intrinsic tyrosine kinase activity of the EGF receptor is required for biological activity (Chenet al., 1987; Honegger et al., 1987b), it hasbeen less clear whether this enzymatic function is required for ligand-induced internalizationand postendocytic targeting (Chen et al., 1989; Felder et al., 1990; Glenney et al., 1988; A
EGF RECEPTOR: HOLO
Mi2?
C1022 CtOZ2
Mi”
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025
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I
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4
8
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FIG. 9. Ligand-induced degradation of EGF receptors. A, Western blot analysisof total cell EGF receptor mass. Cells expressing the indicated receptors were treated without or with 50 mM EGF for 24 h prior to analysis. Receptor mass was determined by densitometry and is expressed as the ratioof EGF-treated to control using the average of duplicate experiments.Arrows indicate the position of’ immunostained mutant EGFreceptors. R,pulse-chase analysis of the effect of EGFondegradation of holo kinase-active (0, 0)and M”c’1022 kinase-inactive (A,A) EGF receptors. Triplicate wells from EGF-treated (0,A) and control (0,A) cultures were measured. Values for t3,>of receptor degradation were: holo receptor 48 and 15 h and M;”c’1022 receptor 52 and 17 h without and with 100 nM EGF, respectively.
Internalization and Down-regulation of the EGF Receptor
11091
Honegger et al., 1987a, 1990). The current study provides receptors that displayed a ligand-induced increase in endoseveral independent lines of evidence that intrinsic tyrosine cytic rates appear able to saturate the endocytic system and kinase activity is required for rapid endocytosis of the holo t o down-regulate (Chen et al., 1989). EGF receptor, but does not regulate recycling or lysosomal Together, these data lead to the following model. In the targeting. absence of tyrosine kinase activity (empty receptors oroccuThere are three possible fates of all cell surface receptors pied receptors modified by site-directed mutagenesis), EGF by random entrapmentin endocytic with respect to coated pit internalization. Receptors can be receptors are internalized a conformation change specifically included in coated pits, they can excluded be from structures. Ligand binding leads to those structures, or they can be neither included nor excluded. that allows interaction with coated pit components. Because In the lastcase, the receptors shouldbe internalized a t a rate ligand binding activates intrinsic protein tyrosine kinase acthat reflects random captureby invaginating endocytic struc- tivity, phosphorylationof some component by the EGFreceptures. This rate can be estimated by considering the fastest tor could stabilize this interaction, leading to the observed reported internalization rates of receptors. If a receptor is high affinity binding to the endocytic apparatus. Receptors 100% captured by coated pits, then its specific internalization modified by site-directed mutagenesiscould bind either more rate will equal that of the coated pit itself (Goldstein et al., tightly or less tightly to coated pits, depending on the struc1984). Carbohydrate-binding receptorshave among the fastest tural consequence of the modification. However, "ligand-ininternalizationrates, usually ranging between 1.2 and 1.3 d u c e d internalization and subsequentreceptor down-regulaaffinity forendocytic rnin", with the fastest reported rate being 4.1 min" (Ward tioncan onlyoccurwhenreceptor structures is significantly increased by occupancy. Since C'andKaplan, 1990; Magnussonand Berg,1989).Because coated pits comprise about 2%of the cell surface, random terminal sequences are required for ligand-induced internalientrapment by coated pits should yield internalization rates zation of EGF receptors (Chen et al., 1989),kinase-active of between 0.03 and 0.08 min". If random captureby smooth receptors which lack these sequences neither undergo ligandpits isincluded, the estimate iseven higher. Recently, specific induced down-regulation norloss of receptor mass. Other investigators have postulated that tyrosine kinase internalization rates of Tf receptors lacking either a cytoplasmic tail (Jing et al., 1990) or a specific internalization activity is required for EGF receptor down-regulation due to sequence (McGraw andMaxfield, 1990) have been estimated, a kinase-mediated block in receptor recycling rather than at yielding values between 0.04 and 0.06 min". Similarly, re- the level of ligand-induced internalization (Honegger et al., placing the cytoplasmic tail of the avian asialoglycoprotein 1987a, 1990; Felder et al., 1990). Despite extensive investigareceptor with unrelated sequences from Xenopus globin pro- tion, we could find noevidence to support thisclaim. Careful, duced receptors with internalization rates between 0.02 and direct measurement of the rate and extent of EGF recycling 0.06 min" (Verrey et al., 1990). Finally, all "internalization- in cells expressingeitherkinase-active or kinase-inactive defective" LDL receptors are internalized at a rate between receptors indicates that EGF recycling is very similar for both 0.02 and 0.03 min" (Davis et al., 1987). Becausethese mutant receptors. Further, cell fractionation and density shift coloLDL receptors are highly mobile in the plasma membrane calization experiments demonstrate no qualitative difference (Barak andWebb, 1982; Goldstein et al., 1984), but are unable between the postendocytic compartmentation of kinase-active t o cluster in coated pits(Beisiegel et al., 1981), their internal- orkinase-inactive receptors.Honeggar et al. (1987a, 1990) ization rate should accuratelyreflect random entrapment by and Felder et al. (1990) also failed to observe kinase-dependcoated pits or other endocytic structures. All these data sug- ent EGF receptor internalization. Analysis of their data regest that the specific internalization rate of randomly enveals thattheinternalizationrate for thekinase-inactive trapped receptors will fall somewhere between 0.02 and 0.06 receptor was between 0.02 and 0.06 min",well within the rnin". This is precisely the range of values we observed for range reported here. However, the apparent internalization kinase-inactivereceptors (-0.03 min"), unoccupiedreceprate of the wild-type receptor in their experiments, was only a cytoplasmic domain. tors, and receptors lacking about 0.10 min" (Honegger et al., 1987a), somewhat below The specific internalization rate of the ligand-bound, kithe range of 0.14-0.5 min" reported for the EGF receptor in nase-active receptorsat low occupancies averaged 0.32 rnin", a wide variety of cell types (Gex-Fabry andDeLisi, 1986; Gill indicating that 32%of the occupied receptors were internal- et al., 1988; Lloyd and Ascoli, 1983; Waters et al., 1990; Wiley ized per minute. This valuedeclined to 0.06 min" as the and Cunningham, 1982). An inspection of their technique for endocytic system became saturated at high levels of receptor measuring internalization rates(Honegger et al., 1987a, 1990) occupancy. In contrast, the internalization rate for ligand- shows that conditions that favor high numbers of occupied occupied, kinase-inactive receptorswas 0.03 min" a t all levels receptors were used. This would bias estimates of specific of occupancy. The low internalization rate of kinase-inactive internalization rates of wild-type receptors to lower values receptors was also shared by EGF receptors lacking a cyto- (Benveniste et al., 1988; Wiley, 1988; Chen et al., 1989; Lund plasmic domain (IS-) and by unoccupied wild type receptors et al., 1990b). In addition, their technique measures net interas measured by antibody internalization. Therefore, thelarge nalization rates (total per cell) rather than specific internalidifferences in endocytic behavior between kinase-active and zation rates (rate per receptor). Net internalization rates of kinase-inactive EGF receptors appear to be explained by the cells undergoingdown-regulation will always fall as a function ability of kinase-activeEGFreceptorstobindwith high of time relative to cells maintaining constantsurface receptor affinity to coated pits or other componentsof the endocytic number, narrowing the initial difference between kinase-acapparatus. EGF receptor truncation mutants ~ ' 1 0 2 2which tive and kinase-inactivereceptors. lack intrinsic tyrosine kinase activity display occupancy-faInterestingly, thepostendocytic trafficking patternof EGF holo, and its receptor appears to cilitatedinternalization t o a lesser extentthanthe be highly cell-type dependent. kinase-active receptor, and they display a relativelylow affin- Although the EGF receptorrecycles efficiently in transfected ity for the endocytic system. However, kinase-active ~ ' 1 0 2 2 B82 cells and other types (Dunn and Hubbard, 1984; Korc EGF receptor mutants have a significantly greater affinityfor and Magun, 1985; Murthy et al., 1986; Sorkin et al., 1989), the endocytic apparatusand undergo internalizationand there appears to be little if any EGF receptor recycling in down-regulation with a corresponding greater efficiency. All humanfibroblasts(StoscheckandCarpenter, 1984). Recy-
11092
Internalization Down-regulation and
of the EGF Receptor
cling of both EGF and itsreceptor is most evident in trans- effects of receptor/ligand recycling anddegradationon k, formed cells which express high numbers of receptors, such values obtained from short-time internalization experiments. as A431 (Sorkin et al., 1991a) or B82. This could be due to The starting point is a kinetic species balance equation for the saturationof sorting endosome components that mediate the numberof intracellular ligand/receptorcomplexes at time lysosomal targeting. Overexpression of proteins normally tar- t, [LR],( t ) as follows, geted to lysosomes has been suggested to saturate the sorting mechanism in the trans-Golgi complex (Williams and Fukuda, 1990), leading to their appearance at thecell surface. Differences in cell function could be responsible for differences in t = 0, [ L R ] ,= 0 the ability of cells to sort materials to the lysosomes. For example, the ratio of diacytosis to lysosomal targeting of where [LR],$is the number of complexes on the surface at endosomal contents variesbetween the apical and basolateral time t, k, is the complex endocytic rate constant, k, is the surfaces of epithelial cells during transcytosis (Bomsel et al., receptor recycling rate constant, and kh is the intracellular 1989). An appealing hypothesis is that cell transformation is degradation rate constant. The solution to this differential equation can be obtained facilitated by a reduced expression of proteins which specifically target tyrosine kinase receptors tolysosomes, since this through a variation-of-parameters approach(see, for example, would also reduce growth factor utilization. Further studies Ritger and Rose, 1968), are needed to critically test this hypothesis. Nevertheless, tyrosinekinaseactivity per se is clearly not requiredfor [ L R ] , ( t= ) ~,,[LR],,(T)~"'.''~'"''~T (-4-2) lysosomal targeting of eitherEGF or its receptor. The M7"c'1022 mutantEGFreceptorlackskinaseactivity, where 7 is a dummy variable for integration. This solution but undergoes accelerated degradation following ligand-in- has a clear physical interpretation. For any number of comduced internalization. Because holo kinase-inactive receptors plexes on the surface at a given time 7, the corresponding undergo constitutive turnover andsince EGF internalizedby number of intracellular complexes at a later time t will be these receptors is degraded, some lysosomal targeting must equal to the product of the internalization rate for those continuously occur in the absence of kinase activity. original cell surface complexes and the "decay" of the intraDifferences in internalization rates between occupied and cellular complexesdue torecycling and/or degradation during empty receptors correlate well with down-regulation. Mutathe intervening timeperiod ( t - T). tions in the EGF receptor that impair occupancy-induced To calculate the precise effect of recycling and/or degrainternalization rates impair occupancy-induced down-reguladation on an estimate for k,, we would need to know the tion, regardless of the receptor domainmodified (Chen et al., entire time-history of the surface complex number [LR],,(t). 1989; Heisermann et al., 1990; Wells et al., 1990). Receptor This requires writing kinetic species balance equations for mutations that enhance internalization rates also enhance that quantity along with all other quantities involved, such as down-regulation (Chen et al., 1989). Phosphorylation of the surface free receptor number, intracellular free receptor numEGF receptor at T h P 4 by protein kinase C simultaneously ber, and extracellular and intracellular free ligand concentrablocksligand-induced internalizationand down-regulation tions. This will be presented elsewhere,2 but more insight can independent of its effect on receptor kinase activity or affinity be gained from an approximate estimate of the effect of (Lund et al., 1990a). Together, all these data indicate that the proximal cause of EGF-induced receptor down-regulation is interest. We note that the predictionsfrom this estimate are conservativewhencompared occupancy-induced endocytosis. This in turnwould lead to an consistentwith,andinfact, increase in receptor targeting to lysosomes and subsequent with the more comprehensive model computation results. Recycling and/or degradation will have their greatest effect degradation by increasing the intracellular pool of receptors on the numberof internalized complexes, [ L R ] ; ( twhen ), that at steady state. Although lysosomal targeting appears to be number is as large as possible. This is because recycling and mediated by differential receptorlocalization within structures of the multivesicular body (Felder et al., 1990; Hopkins degradation processesoccur atratesproportionaltothat number. In our experiments, the number of surface complexes, et al., 1990), this process is most likely proportional to the [ L R ] , ( t ) builds , up from an initial value of 0 at t = 0 to a internal pool of receptors rather than receptor tyrosine kinase quasi-steady statevalue once the receptor/ligand binding and activity. The specific role of tyrosine kinase activity in the overall process of down-regulation appears to be at the level dissociation processes reach a dynamic balance (Wiley and will of stabilizing receptor interactions with coated pits or other Cunningham, 1981). This value is called [LR],,. [LR]i(t) endocytic structures. Because intrinsic tyrosine kinase activ- correspondingly increase from an initial value of 0 as [ L R ] , effects of ity appears to be required for both efficient internalization increases. The value of [LRIi(t),andthusthe and down-regulation of other surface receptors of this class recycling/degradation, will be conservatively overestimated if (Russell et al., 1987; Sorkin et al., 1991b), phosphorylationof we assumethat [ L R ] , ( t )= [LR],, for all t. This is the specific proteins in the endocytic pathway may play an im- assumption made toderive the analytical estimate. With this assumption on [LR],,the solution expressed in portant role in regulatingsignaltransduction at the cell Equation A-2 simplifies to thefollowing. surface.
$'
Acknowledgments-We thank Cheri S.Lazar for expert help, Kirk Lund and Lee Opresko for critical discussions, Cindy Starbuck for performing the computer simulations, Roberto Campos-Gonzonales for the C13 antipeptide monoclonal antibody, Deborah Cadena for the N-20 polyclonal antipeptide antibody, and Jerry Kaplan for the conjugate of HRP and Tf. APPENDIX
The Effect of Ligand Recycling and Degradation on Internalization Analysis-We derive a conservative estimate of the
The Wiley-Cunningham method for producing an "observed value of k, neglects effectsof recycling and degradation during
' C. Starbuck, H. S. Wiley, and D. A. Lauffenberger, manuscript in preparation.
Internalization Down-regulation and short-timeinternalizationexperiments (Wiley andCunningham, 1982), and uses the expression as follows. (kr)obs
=
(A-4)
t
':i:::)dT
In the present case of [LR],,(t) = [LR],,,,this becomes the following.
of the EGF Receptor
11093
receptor because both k , and k,, appear larger in the case of the former. Therefore, the comparative differences between the truevalues of these parametersfor the two receptor types will be at least as great, and likely greater, than thosebetween the observed values. REFERENCES
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