Galactose-specific Recognition System of Mammalian Liver : Receptor Distribution on the Hepatocyte Cell Surface

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1 Published Online: 1 September, 1981 Supp Info: Downloded from jcb.rupress.org on July 21, 218 Glctose-specific Recognition System of Mmmlin Liver : Receptor Distribution on the Heptocyte Cell Surfce DORIS A. WALL nd ANN L. HUBBARD Deprtment of Cell Biology nd Antomy, The Johns Hopkins University, School of Medicine, Bltimore, Mrylnd 2125 ABSTRACT An isolted perfused liver system ws used to study the distribution of siloglycoprotein (ASGP) binding sites on rt heptocyte cell surfces. The number of surfce receptors ws quntitted by monitoring clernce of ' 25 1-lbeled lignds from the perfuste medium under two conditions tht blocked their internliztion : low temperture (<5 C) or brief formldehyde fixtion. The cell surfce distribution of binding sites ws visulized in the electron microscope with either siloorosomucoid covlently coupled to horserdish peroxidse (ASOR-HRP) or lctosminted ferritin (Lc-Fer), both of which were bound with similr kinetics nd to similr extents s ASOR itself. At low temperture or fter prefixtion, ASGP binding sites were present over much of the sinusoidl cell surfce, but were concentrted most hevily over coted pits. Quntittion of lignd distribution t 4 C with Lc-Fer gve n -7-fold greter density of ferritin prticles over coted membrne thn over uncoted regions. We obtined no evidence for grdul movement of ASGP receptors into or out of coted pits within the time-course of our experiments. Finlly, the number nd distribution of cell surfce binding sites ws unffected by previous exposure to ASOR or by inhibition of endocytic vesicle- lysosome fusion nd ASOR degrdtion t 16 C. Desilylted glycoproteins (ASGPs) re removed from the circultion of mmmls by the prenchyml cells of the liver (1). These cells possess cell surfce receptor tht recognizes nd binds molecules hving exposed glctose, N-cetylglctosmine, or glucose residues (1). After binding receptors, ASGPs re internlized vi coted pits nd coted vesicles nd subsequently pper in complex network of tubules nd uncoted vesicles before reching the lysosomes where they re degrded (2-4). In our previous in vivo studies ofasgp internliztion, little evidence ws obtined for binding of the lignd to the heptocyte cell surfce outside ofcoted pits. However, the rpidity with which bound ASGPs entered the cell (5) precluded ny conclusions regrding the locliztion of initil binding sites. Studies of receptor-medited endocytosis in vriety of other systems hve shown tht lignd internliztion cn be inhibited by the use of low temperture, light fixtion before binding, or vriety of inhibitors. Locliztion of epiderml growth fctor THE JOURNAL Of CELL BIOLOGY " VOLUME 9 SEPTEMBER The Rockefeller University Press /81/9/687/1$1. (EGF) nd 2-mcroglobulin (2M) in fibroblsts nd humn crcinom cells under these conditions indictes tht their initil binding to the cell surfce occurs both in coted nd uncoted regions of the plsm membrne (6-1). However, the binding of low-density lipoprotein(ldl)-ferritin t low temperture or fter formldehyde tretment shows stronger preference for coted pits, region of the cell surfce comprising <2% of the totl fibroblst surfce re (11). Becuse internliztion of ll three lignds ppers to occur primrily vi coted pits nd coted vesicles, movement of lignd-receptor complexes into coted pits hs been suggested. In this pper, we report the results of studies on ASGP receptor distribution under conditions tht prevent lignd internliztion. We hve used the isolted, perfused liver system described by Dunn et l. (4), so tht the temperture of heptocytes nd the perfuste composition cn be controlled without disrupting norml cellulr ssocitions. Our fording is tht ASGP binding sites re concentrted predominntly in 687

2 coted pits under ll conditions so fr exmined. A preliminry report of this work hs been published elsewhere (12). MATERIALS AND METHODS Regents Lctoperoxidse (purified grde) nd horse spleen ferritin (A grde) were obtined from ClBiochem-Behring Corp., Americn Hoechst Corp. (LJoll, CA) ; horserdish peroxidse (type VI), 3-mino-1,2,4-trizole, 3,3'-diminobenzidine (grde 1I), polyvinylpyrrolidone (PVP-4, phrmceuticl grde), nd glucose oxidse (type V) were from Sigm Chemicl Co. (St. Louis, Mo.); glutrldehyde ws obtined from EM Sciences (Fort Wshington, P) ; Sprgue- Dwley rts were purchsed from Chrles River Breeding Lbortories, Wilmington, Mss. Isolted Liver Perfusion Recirculting perfusionofrt livers ws performed ccording to the procedure of Dunn et l. (4), using 125 ml of Krebs-Henseleit blnced slt solution contining 2% PVP-4 s the perfuste medium. The livers were perfused vi cnnul inserted in the heptic portl vein. Temperture ws controlled by pumping the medium through condenser mintined t the pproprite temperture by recirculting wter bth. The liver effluent ws collected in beker through which 95% OZ/5% C2 ws pssed vi gs-permeble fibers. Single-pss perfusions were set up in mnner similr to tht for recirculting perfusion except tht the effluent solution ws diverted before reentry into the liver nd fresh solution substituted. Liver temperture ws determined by mesuring the effluent medium s it flowed out of the orgn. Rts were fsted h before scrifice. Binding nd Dissocition of Lignd The preprtion of siloorosomucoid nd iodintion of proteins hve been described previously (2). Lignds were dded to the liver effluent, llowing mximl mixing with the perfuste before it entered the liver. The mounts nd specific ctivities dded re specified in the Results for ech experiment. In some experiments, clcium present in the Krebs-Henseleit perfusion medium (2 mm) ws removed by the ddition of 2.5 ml of.5 M EDTA (finl concentrtion of 1 mm EDTA) plus sufficient 5 N NOH to mintin constnt ph. Becuse ASGP binding is strictly dependent on the presence of clcium (13), this tretment resulted in prompt dissocition of ccessible (i.e., noninternlized) lignd. In protocols where the dissocition step ws followed by subsequent binding of fresh lignd, the dissocited lignd nd chelting gent were first rinsed out with t lest 1 ml of fresh perfuste. Prefixtion Isolted livers were fixed before binding by single-pss perfusion with 15 ml of 2% formldehyde (freshly prepred from prformldehyde) in Krebs- Henseleit/PVP-4 medium t 4 C nd 25 ml/min. Excess fixtive ws then rinsed out by single pss with 1 ml of cold perfuste medium before the orgn ws plced in the recirculting mode for subsequent binding studies. Temperture-shift Experiments Isolted livers were wrmed rpidly from 2-4 C to 3-31 C by single-pss perfusion with 37 C medium tht hd not circulted through the condenser. The temperture ofthe liver effluent could be rised or lowered between 2-4 C nd 3-31 C within min fter temperture shift wsbegun. Switches between 2-4 C nd 16 C were ccomplished by using twotemperture-controlled wter recircultors set t -7 C (for 2-4 C perfusion) nd 12 C (for l6 C perfusion). Preprtion nd Visuliztion of EM Trcers Procedures for the preprtion of the siloorosomucoid-horserdish peroxidse (ASOR-HRP) conjugte nd oflctosminted ferritin (Lc-Fer) hve been described elsewhere (2). "'I-Lc-Fer nd ASOR-HRP, rdioiodinted either in the ASOR moiety only or in both ASOR nd HRP, were clered from the perfusion medium by the isolted liver with kinetics similr to those found for ' 26 1-ASOR. Binding of iodinted ASOR, ASOR-HRP, or Lc-Fer gve similr numbers ofsurfce ASGP receptors per cell (see Results for "5I-ASOR dt). HRP cytochemistry ws performed ccording to the protocol described erlier (2) or slight modifiction of the procedure of Rodewld (14), in which incubtions were for 2 h t 24 C nd 3-mino-1,2,4-trizole (.2 M) ws dded. The fixtion protocol I of Frnke et l. (15) wsused in most Lc-Fer experiments to fcilitte identifiction ofcoted membrne regions. Quntittion of Lc-Fer Binding Lc-Fer prticles were counted t the electron microscope t screen mgnifiction of x 31,5 or from prints t finl enlrgement of x 8,. The sinusoidl region of heptocytes ws scnned by following the profile of the surfce, including microvillr projections, nd counting ll Lc-Fer prticles seen close to membrne bilyer. Prticles not clerly outside of the cell (i.e., those ssocited with grzing sections of the cell surfce tht my hve been locted just inside the membrne) were not counted, to void confusion with endogenous ferritin prticles found free in the cytoplsm of ll heptocytes. Prticles were scored s either () ssocited with uncoted membrne, including microvillr surfces nd smooth vesicle profiles ner the cell surfce, or s (b) ssocited with coted pits or coted vesicle profiles ner the surfce. The ltter ctegory ws undoubtedly underestimted becuse ny membrne region tht ws not obviously coted ws scored s uncoted. Uncoted orcoted vesicle profiles tht were closeto but notclerly contiguous with the surfce membrne were included in the scoring for two resons : () the EDTA dissocition experiments indicted tht t low temperture virtully ll bound lignd (>95%) ws ccessible to the cheltor nd hence t the surfce ; nd (b) our previous work with livers fixed in situ indicted tht --5% of the coted vesicle profiles were not vesicles, but were res of surfce membrne whose continuity with the cell surfce ws not pprent becuse of the plne of section (see reference 2 for ruthenium red experiments demonstrting this). Quntittion of Coted Pits nd Vesicles Estimtion ofthe number ofcoted pits nd vesicles t the sinusoidl surfce ofheptocytes ws done t the electron microscope t screen mgnifiction of x 12,5. Only heptocytes whose entire periphery ws visible nd whose nuclei could be seen were scored in n ttempt to eliminte those in which the plne of section only grzed the cell surfce. Coted vesicle profiles tht were not close to the cell surfce (e.g., those deep within the cytoplsm in the Golgi-lysosome region) were not counted. The frctionl surfce re contributed by coted pits t the sinusoidl surfce ws estimted s follows. Microgrphs of the sinusoidl region of heptocytes were tken t rndom t stndrd mgnifiction of x 1,. Quntittive nlysis ws performed t finl mgnifiction of x 27,. The reltive surfce re ofcoted pits ws determined by usingclibrted set of lines nd recording the type of plsm membrne (uncoted vs. coted) tht crossed the lines. 2 totl crossings were recorded, two of which were over coted pits. A more extensive nlysis ws not performed, becuse ofthe very smll re contribution of these structures. Rther, n upper limit of 2% of the totl sinusoidl surfce ws ssigned to coted pits. RESULTS Clernce nd Metbolism of "' I-Lignds by the Isolted Perfused Liver t 37 C We first exmined ASOR clernce t 37 C to estblish tht the isolted liver ws cpble of norml binding nd metbolism of ASGPs. Clernce of.2 nmol of ASOR t 37 C occurred with tr/2 of 3-5 min nd resulted in the removl of 7% of the totl lignd from the medium by 15 min (Fig. 1). This rte ws somewht slower thn the min found in in vivo studies (2, 15-17), but ws comprble to the results of Dunn et l. (4) with the isolted perfused liver. After the rpid initil decline in circulting rdioctivity, grdul increse ws seen beginning t 15 min fter lignd dministrtion. This rise continued over the next hour nd ws ccompnied by decrese in the percent of cid-insoluble rdioctivity in the perfuste, from 87 to 25%. These clernce kinetics nd lignd solubility chrcteristics indicted tht ASOR ws being internlized by cells in the liver, degrded to low moleculr weight mteril (125 1 nd 1251-monoiodotyrosine ; see reference 3) nd relesed bck into the medium. Low Temperture Perfusion CONDITIONS THAT BLOCKED `26 1-ASOR CLEAR- ANCE : To loclize the initil ASGP binding sites on heptocyte cell surfces, it ws necessry to first estblish conditions 688 THE JOURNAL OF CELL BIOLOGY " VOLUME 9, 1981

3 E 7 r?. x 6 E c -"- 5 4 U 3 w 2 F- Q L 1 w W o_ I I I 1 I I I I I TIME (minutes) 8 F U Q 6 WJm J 4 z U 2 e FIGURE 1 Clernce of "'I-ASOR by n isolted perfused liver mintined t 37 C. The liver ws llowed to equilibrte for 15 min t 37 C before the ddition of.2 nmol (2.1 x 16 cpm) of 12s1- ASOR to the perfusion medium..5-ml liquots were tken t the indicted times, nd rdioctivity in ech smple ws counted. Ech liquot ws cooled on ice before n equl volume of cold 1% TCA ws dded. After t lest 1 h, the precipittes were centrifuged (15 min t 3, rpm), nd the superntes nd pellets were seprted nd counted. The increse in perfuste rdioctivity strting t 15 min ws ttributble predominntly to cid-soluble mteril s evidenced by decrese in the percentge of cid-insoluble rdioctivity from 87% t 1 min to 25% t 3-9 min. tht llowed lignd binding but blocked lignd internliztion. When the isolted liver ws cooled to temperture of 2-5 C, the clernce kinetics of 1251-ASOR (.1 nmol) slowed to rtes one third to one fourth of those seen t 37'C (see Fig. 2). In ddition, no decrese in cid-insoluble rdioctivity fter the initil decline ws seen t this temperture, even fter 2 h of incubtion. Furthermore, the ddition of EDTA (finl concentrtion 1 mm) to remove ll free clcium from the perfusion medium resulted in prompt rise of rdioctivity in the perfuste bck to the pek level seen fter the initil ddition of lignd (Fig. 2). Finlly, ll ofthe relesed 1251-lignd ws cidinsoluble. These results indicted tht the ASOR tht ws clered from the perfuste t 2-5 C hd remined on the cell surfce without internliztion or subsequent degrdtion by heptocytes. The binding of lignd t low temperture ws quite stble. Livers to which either.22 or 2.2 nmol of ASOR ws first bound for 6 min t 2-5 C were rinsed free of excess unbound lignd nd then incubted for n dditionl 6 min ech, first in the bsence of circulting lignd, then in the presence of2.2 nmol of unlbelled ASOR. Finlly, sufficient EDTA ws dded to remove free clcium nd dissocite ccessible ASOR. Perfuste rdioctivity ws monitored throughout nd no detectble loss of bound ASOR from the cell surfce occurred until the finl EDTA step, t which time virtully ll of the rdioctivity tht hd originlly been clered from the circultion ws relesed bck into the perfuste (dt not shown). ESTABLISHMENT OF BINDING CONDITIONS REQUIRED TO SATURATE CELL SURFACE RECEPTORS : We next determined the conditions t 4 C necessry to sturte the vilble ASGP binding sites to ensure tht we were visulizing the mjority of ASGP receptors in our EM locliztion experiments. The time-course of ASOR-HRP clernce from the perfuste is shown in Fig. 3. Similr results were obtined when ASOR ws used s the lignd. An initil rpid phse of binding ws followed by grdul decrese to plteu t round 2 h. In severl experiments, the decrese in circulting ASOR-HRP between 1 nd 2 h verged % ofthe totl mount of ASOR-HRP bound over the 2-h period. A 1-h incubtion ws chosen s stndrd nd more convenient time } 5 u 4 3 rr w ASOR ASOR CLEARANCE CLEARANCE 5L _L ?LO I5 1 -L TIME (minutes from strt of ech phse) FIGURE 2 Effect of low temperture on clernce of "'I-ASOR by the isolted perfused liver. After the liver ws equilibrted for 15 min t 37 C,.1 nmol (3.2 x 16 cpm) of 1251-ASOR ws dded to the perfuste, nd.5-ml liquots were tken t the indicted times. After 1 min, the liver ws cooled to 5 C nd similr liquot of "'I-ASOR ws dded. Clernce ws monitored for 3 min, then EDTA ws dded to dissocite ccessible lignd, nd liquots were tken over the next 25 min. Removl of clcium by EDTA resulted in prompt dissocition of the bound ASOR, indicting tht internliztion of the lignd hd not tken plce.,..8r 37~ 125r nc k, 4' ASOR-HRP CLEARANCE 1 W - ( ~-/< I I I TIME (minutes from strt of ech phse) FIGURE 3 Time-course of 1251-ASOR-HRP binding to the isolted perfused liver t 2-5 C. After equilibrtion for 15 min t 37 C,.2 nmol (2.1 x 16 cpm) of 1251-ASOR ws dded to the perfuste, nd the incubtion continued for 1 min, t which time the liver ws cooled to 4 C nd fresh perfusion medium ws dded. 2.2 nmol (4.8 x 15 cpm) of 1251-ASOR -HRP (lbeled in the ASOR moiety only) plus.2 nmol of 1251-ASOR ws dded, nd clernce ws monitored for 12 min. Clernce t low temperture shows rpid initil decline, then levels off to pproch plteu between 6 nd 12 min. The ddition of EDTA t 4 C resulted in prompt relese of lignd bck into the perfuste. The extent of decrese in circulting rdioctivity between 6 nd 12 min in this experiment ws somewht greter thn the usul 15-2%. The liver weighed 9.21 gm. WALL AND HUBBARD Receptor Distribution on Rt Heptocyte Surfces 689

4 for subsequent binding nd locliztion studies t the low temperture. We then exmined the effect of dded lignd concentrtion on the mount of ASOR bound by the liver in 6 min t 2-4 C. The results re presented in Fig. 4. Becuse the ctul weight of the liver used in these experiments ws not known until the end of the experiment, the mount of ASOR bound is expressed s the estimted number of surfce receptors per cell, nd the mount of ASOR dded s rtio of ASOR input to liver weight. Although the mount of lignd bound incresed rpidly t low levels of dded ASOR, binding begn to pproch plteu of 3, surfce receptors per cell t -4.4 nmol of dded lignd (rtio: 2-3). Mny ssumptions re inherent in the clcultion of this figure, including the following: () tht 1 g of liver, wet weight, contins 1.38 x 18 prenchyml cells (18) ; (b) tht the moleculr weight of ASOR is 45,, of ASOR-HRP is 9,, nd of Lc-Fer is 46,; (c) tht ll lignd clered ws specificlly bound ; (d) tht only heptocytes, nd not endothelil or Kupffer cells, bound the lignds ; (e) tht one receptor bound one lignd molecule, despite the multivlency of the lignd ; nd (f) tht ll regions of the liver were uniformly perfused. Becuse of these uncertinties, the ctul numbers of receptors per cell cn only be regrded s n estimte. Nonetheless, the vlues obtined were quite consistent in mny different experiments using severl different preprtions of lignd. The mjority of experiments to be described below were performed with 2.2 nmol of ASGP dded for 6-min incubtion time. Becuse 2.2 nmol of dded lignd resulted in 7-8% ofmximl binding t 6 min (Fig. 4 : the drk br on the J J U F U I RATIO ASOR INPUT/LIVER WEIGHT FIGURE 4 Effect of lignd concentrtion on the binding of ASOR to the isolted perfused liver. The mount of ASOR bound is expressed on the ordinte s the estimted number of receptors per cell nd the mount of ASOR dded s the rtio of!1g of ASOR input to the wet weight of the liver in grms. The drk br indictes those rtios obtined with the ddition of 2.2 nmol of ASOR, the stndrd lignd level used for most experiments. ' 25 1-ASOR (.1 nmol) plus vrying mounts of unlbeled ASOR ( nmol) ws dded to the perfuste of isolted livers nd llowed to circulte for 6 min t 4 C. EDTA ws then dded to finl concentrtion of 1 mm to ensure tht ll lignd ws t the cell surfce. In some experiments, dissocited lignd nd EDTA ws rinsed out nd further rounds of binding were performed on the sme liver (these points indicted by different open symbols for ech liver). Filled symbols represent points derived from single rounds of ASOR (circles) or Lc-Fer (squres) binding, ech to different liver. Sturtion of surfce binding ws not complete until the mount of lignd dded ws t lest 4-5 nmol (rtios of 2 or more), giving sturtion level of =3, receptors/cell. v bciss indictes those experiments in which 2.2 nmol oflignd were dded), nd becuse 6-min incubtion gve 8-85% of the mximl binding t 12 min (Fig. 3), we estimted tht we were visulizing 55-7% of the totl cell surfce receptors in our EM locliztion experiments. LOCALIZATION OF ASOR-HRP BINDING : Hvingestblished the conditions t 4 C resulting in mximum binding of ASOR nd ASOR-HRP without internliztion, we next used the cytochemicl trcer ASOR-HRP to visulize the ASGP binding sites on heptocyte cell surfces. Incubtion of livers t 2-5 C with 2.2 nmol of ASOR-HRP for 6 min, followed by processing of the tissue for the detection of HRP ctivity, reveled nonuniform distribution of HRP stining on heptocyte cell surfces (Fig. 5A nd B). ASOR-HRP ws concentrted in pits of 9-35 nm length t the surfce of the cells, often t the bse ofmicrovilli. The rest of the cell surfce ws lightly stined in mny, but not ll, regions of the tissue. Stined pits were occsionlly seen on the lterl surfce of heptocytes. Not ll pits contined lignd, nd stined pits could occsionlly be found in close proximity to unstined ones (Fig. 5A). Both the drk stining of pits nd the lighter stining outside were eliminted when the liver ws exposed to 1 mm EDTA for 1 min before fixtion (Fig. 5 D), indicting tht the stining observed ws result of ASOR-HRP binding to the cell surfce. Kupffer nd endothelil cells lso showed clcium-dependent stining ofpits in ddition to light stining of the cell surfce. Becuse we were concerned tht some of the observed binding might be nonspecific, especilly the low level seen outside of coted pits, number of controls were performed. () Addition of 6-fold excess ofasor, together with ASOR or 1251-ASOR-HRP, drsticlly reduced clernce of the rdiolbeled lignds from the perfuste medium, nd eliminted both the hevy HRP stining of pits nd the light stining of the rest of the heptocyte cell surfce. (b) In contrst, similr excess of fully silylted orosomucoid (OR) or mnnn ( mnnose-terminting glycoprotein tht inhibits binding to endothelil nd Kupffer cells) hd little effect on clernce from the perfuste or distribution ofasor-hrp on heptocyte cell surfces, lthough mnnn reduced the stining observed on endothelil nd Kupffer cells. Exposure of livers to low levels of ASOR-HRP (.22 nmol) resulted in <2% of mximum binding. Nonetheless, the sme distribution of binding sites s tht seen t higher levels (2.2 nmol of ASOR-HRP) ws found, suggesting tht binding sites both inside nd outside of pits hve similr ffinities for the lignd. EXAMINATION OF ASOR-HRP DISTRIBUTION AFTER DIFFERENT INCUBATION TIMES : To determine whether slow movement of bound lignd ws occurring during the 6- min incubtion t 2-5 C, we exmined ASOR-HRP distribution fter shorter incubtion times. Binding of 2.2 nmol of ASOR-HRP for 2 (Fig. 5 C) or 4 min resulted in generlly lighter stining both inside nd outside of pits, with no obvious chnges in the reltive stining intensity ofthe two res. Two ttempts t binding for shorter times did not give sufficient stining for cler visuliztion of the lignd. Next, ASOR-HRP ws bound for 6 min t the low temperture, excess unbound rinsed out, nd the livers incubted for n dditionl 6 min t low temperture before fixtion. The sme result ws obtined s when livers were fixed immeditely fter the initil 6-min binding. Tht is, regions were seen in which the entire cell surfce ws lightly stined, while surfce 69 THE JOURNAL OF CELL BIOLOGY " VOLUME 9, 1981

5 FIGURE 5 Electron microgrphs of the peripherl cytoplsm of heptocytes in livers fixed fter incubtion with 2.2 nmol ASOR- HRP t 2-5 C. In ll experiments, excess unbound lignd ws rinsed out before the liver ws fixed nd processed for HRP cytochemistry. In A, 8, nd D, ASOR-HRP ws bound for 6 min ; in C, the liver ws rinsed nd fixed 2 min fter lignd dministrtion. Although HRP ctivity is concentrted in pits, light stining is seen over most of the cell surfce (most redily seen in A nd C, nd questionble in 8). The stining outside of pits is prticulrly evident in res where grzing sections pss cross the periphery of microvillr projections, s in the centrl portion of A. The peroxisome seen in 8 (Ps) stins hevily becuse the ctlse ctivity of these orgnelles ws not inhibited by minotrizole in this experiment. The liver shown in Dws treted with 1 mm EDTA fter the ASOR-HRP incubtion. This tretment removed ll surfce ctivity, both inside nd outside of pits. In A, hevily stined pits re seen in close proximity to unstined ones (unstined pits mrked with rrows in A). In C, smoothsurfced membrne vesicles nd tubules (SM) re prominent in the heptocyte periphery. A, C nd D : x 5,. Br,.25 gm. 8, x 8,. Br,.2,m. pits showed hevy deposits of HRP rection product. This result rgues ginst grdul shift of lignd molecules into pits from the lightly stined res. LOCALIZATION OF LAC-FER BINDING : QUANTITATION OF LIGAND DISTRIBUTION : Although the ASOR-HRP conjugte ws extremely useful s n indictor of overll distribution of ASGP binding sites, this lignd did not llow the binding to be redily quntitted. In ddition, we were not ble to visulize coted membrne regions fter HRP cytochemistry ws performed. Therefore, we turned to the use of the prticulte trcer Lc-Fer to estimte the reltive numbers of ASGP receptors ssocited with coted vs. uncoted membrne regions. Binding of Lc-Fer (8 nmol for 6 min or 14 nmol for 125 min) t 2-5 C reveled similr distribution of ASGP binding sites s the ASOR-HRP experiments. 58% of the totl Lc- Fer prticles counted (362 out of 628 totl : counts from two experiments were similr nd were therefore pooled) were ssocited with coted membrne regions which comprise <2% of the totl surfce membrne re (see Fig. 6). The remining 42% of Lc-Fer ws ssocited with uncoted membrne, mking the concentrtion of Lc-Fer over coted regions t lest 67-fold higher thn the concentrtion over uncoted membrne. Becuse coted pits were often difficult to identify with certinty in the Lc-Fer experiments, we consider this vlue (67-fold higher) to be lower limit of ASGP concentrtion in coted pits of heptocytes. Using the dt from these morphologicl experiments, the number of Lc-Fer prticles bound per heptocyte ws clculted to be 5,4 to 12,. 1 This vlue is pproximtely equl to 5% of tht obtined from biochemicl studies with Lc- Fer (see Fig. 4). Two fctors re known to contribute to this discrepncy : () binding of Lc-Fer to sinusoidl lining cells, which could ccount for no more thn 25% of the totl Lc- Fer bound, ccording to ASOR competition experiments (not shown); (b) loss of 4-5% of the bound Lc-Fer during processing of the tissue for EM. With these corrections, we still obtin lower estimte of Lc-Fer prticles bound using morphologic techniques (16% of tht expected from biochemicl dt). However, other fctors tht re more difficult to 1 This number ws obtined using 17.7 pm for the dimeter of heptocytes, section thickness of 75 nm (25-32 sections/cell), n verge of 5-11 coted pit profiles for heptocyte per section, n verge of two Lc-Fer prticles/pit profile nd 42% of totl Lc-Fer bound outside of pits. WALL AND HUBBARD Receptor Distribution on Rt Heptocyte Surfces 691

6 FIGURE 6 Electron microgrphs of the peripherl cytoplsm of heptocytes in livers fixed fter incubtion with Lc-Fer (8 nmol) for 6 min t 4 C. Excess Lc-Fer ws rinsed out with 4 C perfusion medium before the tissue ws fixed by procedure I of Frnke et l. (15). Binding of Lc-Fer occurred preferentilly in coted pits (indicted by rrowheds : 58% of totl prticles counted). Ferritin prticles ssocited with uncoted membrne re indicted by smll rrows. An sterisk indictes coted pit in which no clerly discernible ferritin prticles re found. The number of Lc-Fer per pit is higher in these selected microgrphs thn the verge vlues seen. However, it should be noted tht empty pits contributed to the low verge vlue of two Lc-Fer per pit used in the quntittion of Lc-Fer binding. A, x 7, ; B-D x 8,. Brs,.2 pm. quntitte lso contribute to n underestimtion by morphology : () uncertinty in the identifiction of some Lc-Fer prticles becuse of their poor contrst ginst the bckground mteril ; (b) elimintion from quntittion of ny Lc-Fer prticles not clerly seen to be on the outer spect of membrne cross-section ; (c) underestimtion of the number of coted pit profiles per section becuse of the difficulty in recognizing cots in some res. Tking ll of these fctors into considertion, the quntittion of Lc-Fer binding site concentrtion in coted pits must be interpreted with cution, becuse the possibility of selective loss exists. Nevertheless, the similrity in binding pttern of ASOR-HRP in pits of similr size nd distribution s coted pits reinforces the Lc-Fer dt. Lignd Distribution fter Prefixtion Incubtion of cells t low tempertures ppers not to prevent movement of some membrne molecules within the bilyer (19). Therefore, the clustering of ASGP receptors in coted pits tht we observed t low temperture my hve resulted from movement induced by lignd binding. We were therefore prompted to exmine other methods for blocking lignd uptke to reinforce our conclusions from the low temperture experiments. The use of formldehyde fixtion ws suggested by the results of Willinghm et l. (8) in which 2M receptors showed less clustered distributionfter brief fixtion thn they did t 4 C. CONDITIONS THAT BLOCKED LIGAND UPTAKE : Abrief tretment by single-pss perfusion of 2% formldehyde in Krebs-Henseleit perfusion medium t 4 C ws found sufficient to completely block internliztion of ASOR t 37 C (see Fig. 7). This is, exposure of prefixed liver to ASOR t 37 C, followed by subsequent exposure to EDTA, resulted in complete dissocition of bound lignd. In contrst, exposure of control livers to ASOR t 37 C resulted in clernce nd degrdtion (Fig. 1). In ddition, prefixtion hd no effect on the extent of binding (i.e., the numbers of cell surfce receptors per cell determined t 2-5 C before or fter formldehyde tretment) or kinetics of binding t either 37 or 2-5 C. These results indicted tht formldehyde prefixtion blocked internliztion (even t 37 C) nd could therefore be used to visulize representtive popultion of ASGP receptors t the heptocyte cell surfce. However, the EM locliztion experiments were performed t low tempertures becuse morphologicl dmge occurred if formldehyde-treted livers were subsequently wrmed t 37 C. LOCALIZATION OF ASOR-HRP BINDING AFTER PREFIXATION : Binding of 2.2 nmol of ASOR-HRP for 6 min t 2-5 C to formldehyde-treted livers reveled distribution of ASGP binding sites tht ws essentilly identicl to tht seen t low temperture without prefixtion (compre Fig. 8 with Fig. 5A-Q. The trcer ws gin concentrted in pits t the cell surfce, with light stining of the surfce outside of pits in mny res. Other Approches to Blockge of Lignd Internliztion We hve ttempted to inhibit endocytosis using, in ddition to low temperture nd prefixtion, vriety of regents reported by others to be effective. To dte, of 1-15 mm methylmine, 1 mm phenylglyoxl,.4 mm N-ethylmleimide, 6 ItM dnsylcdverine, nd 1 mm NF plus 1 mm KCN, none hs prevented ASOR internliztion. We re continuing 69 2 THE JOURNAL OF CELL BIOLOGY " VOLUME 9, 1981

7 to investigte the use of these nd other inhibitors in our study of ASGP endocytosis. Modultion of ASGP Receptor Activity nd Effects on Cell Surfce Receptor Number nd Distribution The locliztion experiments described bove were performed on livers tht hd not been previously exposed to ASGPs under conditions llowing binding nd internliztion (i.e., elevted tempertures). Prior physiologicl exposure of ech liver to circulting ASGPs ws therefore n unknown vrible. Becuse ctive internliztion of ASGPs might be expected to influence the disposition of these receptors within the vrious subcellulr comprtments, including the cell sur- =e x 6 E U Q ' ASOR I-ASOR CLEARANCE LEFORE FIXATION CLEARANCE. AFTER FIXATION c II I I I 1I -I I // r 2 I TIME (minutes from strt of ech phse) FIGURE 7 Effect of formldehyde prefixtion of clernce of ASOR by the isolted perfused liver. The liver ws llowed to stbilize t 37 C for 1 min before.1 nmol of ' 26 1-ASOR (1.2 x 16 cpm) were dded to the perfuste. After monitoring clernce for 1 min, we prefixed the orgn t 4 C by single-pss perfusion with 15 ml of 2% formldehyde in Krebs-Henseleit medium. Fixtive ws rinsed out, nd the liver wrmed to 37 C before second ' 25 1-ASOR clernce ws performed exctly s before. After the end of the second clernce period, EDTA ws dded to 1 mm to dissocite surfce-bound lignd. Prefixtion hd no effect on the kinetics of clernce, but the EDTA dissocition indicted tht ll of the bound ASOR ws still t the cell surfce. fce, we investigted the effects of receptor ctivity on the number nd distribution of ASGP cell surfce binding sites. EFFECT OF PREEXPOSURE TO ASOR ON CELL SURFACE ASGP RECEPTORS : First, when n excess (4.4 nmol) of unlbeled ASOR ws perfused through liver for 2 min t 37C before glutrldehyde fixtion nd processing for optiml visuliztion of coted pits, no increse ws observed in the number of coted pits or coted vesicles found in exposed livers when compred to controls perfused 2 min t 37C without dded ASOR (see Mterils nd Methods for the method of scoring used). Therefore, it ppers tht the process of ASGP internliztion does not induce mrked increse in the frequency of these structures. Second, the distribution of ASOR receptors ppered not to be influenced by previous internliztion of ASOR, becuse deliberte introduction of the lignd t 37C before cooling nd ASOR-HRP binding nd visuliztion, resulted in surfce stining qulittively identicl to tht of unexposed cells. This distribution ws obtined whether the livers were preexposed to trce mounts of ASOR (.1 nmol or 5,9 molecules/cell for 1 min t 37C) or to mounts 8.7-fold in excess over the number of cell surfce receptors, ssuming 3, receptors/cell (4.4 nmol or 2.6 x 1 6 molecules/cell for 3 min t 37C). In the ltter experiment, 27% of the dded lignd (72, molecules/cell or 2.3 times the number of receptors t the cell surfce) ws clered from the medium. Neither form of ASOR preexposure ltered the number or distribution of cell surfce receptors. TEMPERATURE-SHIFT EXPERIMENTS : We next exmined the distribution nd number of surfce binding sites in livers tht were sturted with ASOR t low temperture nd then wrmed to induce synchronous internliztion of the lignd. These experiments lso tested the cpcity of lignd bound in nd out of coted pits t low tempertures to be subsequently internlized. 2.2 nmol of " 5 I-ASOR were bound for 6 min t 2-5'C, excess unbound lignd ws rinsed wy, nd the liver ws wrmed to 31'C for 8 min before being returned to low temperture for EDTA tretment. After the wrm-up, 85-9% of 125 I-ASOR ws no longer dissocible by EDTA, indicting tht most of the lignd ws initilly bound to the cell surfce vi functionl receptors cpble of rpidly internlizing the molecules bound to them. In seprte experiment, we found tht 63% of the bound lignd ws no longer FIGURE 8 Electron microgrphs of the peripherl cytoplsm of heptocytes in livers fixed fter formldehyde prefixtion nd ASOR-HRP binding t 4'C. 2.7 nmol of ASOR-HRP were dded to the perfuste of liver tht hd been prefixed by single-pss perfusion with 2% formldehyde. HRP ctivity cn be detected over most of the cell surfce, but is much more concentrted in pits thn in other res. The drk stining of mitochondri) (Mt) criste results from the ctivity of cytochrome oxidse. (A) A series of drkly stined surfce pits re seen long the sinusoidl surfce of n heptocyte. x 5,. Br,.25 ttm. (8) Smooth-surfced membrne vesicles nd tubules (SM) re pprent in the peripherl cytoplsm of heptocytes. x 8,. Br,.2 gm. WALL AND HUBBARD Receptor Distribution on Rt Heptocyte Surfces 693

8 ccessible to EDTA dissocition fter 3.5-min wrm-up, giving t1/2 for internliztion of <3.5 min (Tble I). Furthermore, no significnt chnge could be detected in the number of surfce receptors during ctive internliztion. This is, cells tht hd previously been exposed to 2.2 nmoles of ASOR t 4 C for 6 min, wrmed to 31 C for 3.5 min, cooled, exposed to EDTA, nd then rechllenged with ASOR, bound 225, molecules of lignd per cell, s compred with 179, before wrming. A similr result ws obtined when ASOR binding ws done t low lignd levels (Tble I). The constncy of cell surfce receptor number fter one round of lignd binding nd internliztion prompted us to compre the distribution of the new binding sites fter such exposure to tht found in control livers (i.e., unexposed cells). An experiment ws performed in which 2.2 nmol ofasor ws dded t 4 C for 6 min, the lignd ws rinsed out, nd the liver ws wrmed to 31 C for 4.5 min nd then incubted with 2.2 nmol of ASOR-HRP for 6 min t 4 C. In this cse only those cell surfce receptors tht hd been vcted or tht hd ppered on the surfce during the wrm-up would be visulized, becuse the cytochemicl trcer ws dded only during the second incubtion. Nevertheless, the distribution of ASOR- HRP on the heptocyte surfce ws the sme s tht seen t 4 C with no wrm-up, i.e., light stining of HRP rection product over most of the surfce nd its concentrtion in pits. We therefore obtined no evidence for trnsient ltertions in receptor number or disposition in the membrne during ctive internliztion of siloproteins. Nevertheless, it should be noted tht our binding conditions were substurting, mking precise determintion of ctul receptor number difficult. 16 C PERFUSION : QUANTITATION OF ASGP RECEPTORS ON THE CELL SURFACE DURING INTERNALIZATION IN THE ABSENCE OF DEGRADATION : Temperture mnipultion cn be used to block certin steps in the endocytic process occurring fter lignd internliztion. Dunn et l. (5) hve recently shown tht livers mintined t 16 C will internlize ASGP but not degrde it, becuse of filure of endo- TABLE I Effect of Lignd Internliztion on Subsequent Binding to the Cell Surfce ASOR dded$ nmol nmol molecules/cell ASOR bound or internlized Percent of totl ASOR bound or internlized First binding , , 74 Internlized , 63 during , 71 wrm-up Second bind , 35 ing , 74 "` The experimentl protocol ws s follows: () bind 2.2 or.23 nmol '251- ASOR (3.2 x 15 cpm of "'l-asor plus 1 fg or 1 pg unlbeled) t 4 C for 6 min ; (b) rinse out excess lignd with fresh medium ; (c) wrm to 31 C for 3.5 min, then switch bck to 4 C; (d) dissocite ccessible lignd with EDTA; (e) rinse out relesed lignd nd EDTA with fresh medium ; (f) bind ASOR s in step. $ 2.2 nd.23 nmol represent 2.1 nd.24 times the number of cell surfce receptors (ssuming 3, surfce receptors/cell) becuse the liverweights were 15.2 nd 13.7 gm, respectively. Percent of totl bound tht ws internlized during 3.5-min wrm-up to 31 C. cytic vesicles to fuse with lysosomes. We therefore used 16'C perfusion to investigte whether inhibition of the delivery of lignd to lysosomes would lter the number or distribution of cell surfce ASGP receptors. An isolted perfused liver mintined t 16 C ws llowed to internlize ASOR t very high lignd concentrtion (13.3 nmol of ASOR dded) for 245 min, t which time 16% of the circulting lignd (78, molecules/cell) ws clered from the perfuste, nd the decline in perfuste rdioctivity pproched plteu. The liver ws then rpidly cooled to 4 C, nd ny remining surfce-bound ASOR ws removed with EDTA nd rinsed out, together with unbound lignd. ASOR- HRP (2.2 nmol) plus trce mount of ASOR (.13 nmol) ws then bound for 6 min t 4 C. The number of cell surfce receptors ws mesured t 22,/cell, which ws similr to the numbers obtined in untreted controls (15,- 25,/cell). This result suggests tht involvement of the lysosoml comprtment is not essentil to receptor replenishment on the cell surfce. DISCUSSION ASGP Receptors Are Concentrted in Coted Pits in the Absence of Internliztion The work reported in this pper ws undertken to investigte the distribution ofasgp receptors on the heptocyte cell surfce in the bsence of lignd internliztion. Both low temperture nd brief formldehyde fixtion were used to block endocytosis, nd both reveled the sme pttern : ASGP binding sites were concentrted preferentilly in coted pits but, in mny res, were lso present over the rest of the cell surfce.' Nonrndom distribution of cell surfce receptors hs been described in other systems. In cultured humn fibroblsts, LDL (11, 2, 21), 2M (8, 9), nd EGF (6) llshowed some preference for binding to coted membrne regions t -4 C. However, in none of these cses ws the specificity for coted membrne bsolute. Tht is, binding of lignds lso occurred outside of these specilized res of the plsm membrne. In the sme studies, quntittion of surfce binding sites reveled tht the mount oflignd ssocited with coted pits ws quite vrible: from 1% of the totl bound 2M, yielding five times greter concentrtion in coted pits thn over the remining surfce (ssuming the former to be 2% ofthe totl cell surfce re), to 34% of EGF (25 times greter) to 49-83% with LDL ( times greter). In our experiments, 58% of the Lc-Fer prticles were ssocited with coted pits, giving 67 times greter concentrtion over these regions thn over the remining cell surfce. The results of prefixtion studies on fibroblst surfce receptor distribution vried with the lignd used. Formldehyde tretment (2.5% in phosphte-buffered sline t 4 C for 45 min) did not chnge the LDL-ferritin distribution from tht seen t 4 C without fixtion (11), but reduced the clustering of 2M receptors into coted pits (8;.2% HCHO in phosphtebuffered sline t 23 C for 5 min). Becuse fixtion with formldehyde (2% HCHO in Krebs-Henseleit/PVP 4 t 4 C for 6 min) hd no discernible effect on ASOR-HRP distribution 'For purposes of discussion, we will ssume tht the ASGP receptor is responsible both for initil binding of the lignd to the cell surfce s well s for its subsequent internliztion, nd use the terms "binding site" nd "receptor" interchngebly. 694 THE JOURNAL OF CELL BIOLOGY " VOLUME 9, 1981

9 t the heptocyte cell surfce, our results most closely prllel those obtined in the LDL receptor studies. However, Ziomek et l. (19) hve shown, by vrying the concentrtion of fixtive, tht the movement of integrl membrne proteins in isolted mouse intestinl cells is slowed only t HCHO concentrtions >4% (t C for 15 min). In the bsence of such titrtion studies, it is difficult to know whether the vribility of receptor distributions in different systems is result of the considerble vrition in fixtion conditions. An ttempt t incresing the extent of fixtion in our system by rising the temperture of the formldehyde solution resulted in loss of ASGP binding ctivity. The effect of incubtion time on the observed lignd distribution is of interest becuse in ll locliztion studies the binding times used hve generlly been quite long (2 h t 4 C for LDL-ferritin [11] nd 2M-ferritin [8], 4 min t 4 C for EGF-ferritin [7]). 2 min ws the shortest binding time tht llowed visuliztion of HRP ctivity in our experiments. However, stining intensity cn vry from experiment to experiment, rising the possibility tht shorter incubtion times could be exmined. Nevertheless, no difference in ASOR-HRP distribution ws seen when incubtion times with the lignd vried from 2 min to 2 h. From these dt we cn not sy whether concentrtion of the ASGP receptor in coted pits in the bsence of internliztion occurs before or fter the lignd ttches. In the former cse, concentrtion into pits could occur either by lterl movement of membrne molecules fter their rndom insertion into the bilyer, or by preferentil insertion of receptor molecules into coted membrne regions. In the ltter cse, movement of the lignd-receptor complex would hve to occur t low temperture nd fter formldehyde fixtion to be consistent with our observtions. This possibility is suggested by the results of Ziomek et l. (19) on locliztion of lkline phosphtse nd leucine minopeptidse in isolted mouse intestinl cells. These integrl membrne glycoproteins undergo redistribution by lterl diffusion fter cell dissocition, t either 4 C or fter fixtion with.5-3% formldehyde. However, if lignd-induced redistribution did occur, it is not cler why frction of the occupied ASGP receptors would persist long uncoted regions of the sinusoidl membrne, even fter long incubtions t 4 C. Coted Pits My Be Specilized for the Uptke of Different Lignds The ppernce of unstined pits in close proximity to stined ones reinforces similr observtions mde in our previous in vivo experiments, nd suggests tht coted membrne regions my be specilized for the uptke of different lignds. Nevertheless, it hs been shown tht 2M nd vesiculr stomtitis virus cn enter fibroblsts together within the sme coted pits (1). If this finding cn be generlized to the heptocyte, it suggests tht the concept of one receptor type per pit does not pply. Consistency of Cell Surfce Receptor Number. Receptor Reutiliztion Severl reports on the dynmics of the ASGP receptor hve suggested tht this membrne glycoprotein is spred degrdtion while the lignd it binds is ctbolized within lysosomes. For instnce, determintion of the ASGP receptor t1/2 in Vivo by Tnbe et l. (22) yielded vlue of -88 h, either in the presence or bsence of lrge excess of circulting ASOR. Furthermore, in 18 min t 37 C, isolted heptocytes treted with cycloheximide to block protein synthesis were cpble of binding nd metbolizing 34 times the cell surfce binding cpcity (or twice the totl cellulr binding cpcity, if both internl nd externl receptors re included in the clcultion). In the bsence of cycloheximide, the cells metbolized 4 times the totl cellulr binding cpcity. Such observtions rgue strongly for ASGP receptor reutiliztion. The subcellulr comprtments involved in this reutiliztion process re not known. At one extreme, the ASGP receptor my never leve the cell surfce. At the other, it my enter the cell still complexed with its lignd nd be processed through vrious subcellulr comprtments by n s yet unidentified slvge pthwy before returning to the plsm membrne. In our temperture-shift experiments, the number of ASGP receptors present t the cell surfce ppered to be quite constnt even when heptocytes were ctively engged in ASGP internliztion. Hence, the sme number nd distribution of receptors were seen fter binding sites were sturted nd the bound lignd ws llowed to enter the cell during brief wrm-up. This result demonstrted tht the cell mintined numericlly constnt popultion of ASGP receptors t its surfce. At lest three lterntives could be responsible for this phenomenon : () the receptor could dischrge its lignd to n intrcellulr pool without leving the surfce, (b) the receptor-lignd complex could be internlized nd the sme receptor molecule returned to the surfce fter lignd dissocition, or (c) the complex could be internlized nd replced by different receptor molecule. The recent results of Stockert et l. (23) suggest tht the sme receptor molecule is reutilized, result consistent with lterntive or b. These investigtors reported tht isolted heptocytes whose ccessible ASGP receptors were ltered by neurminidse tretment mintined popultion of modified receptors t the cell surfce. This occurred despite concurrent endocytosis of 2 times the surfce binding cpcity of desilylted ovine submxillry mucin (n N-cetylglctosminyl-terminting glycoprotein tht is still recognized by the neurminidse-treted receptor). Thus, even though only 5-1% of the totl binding cpcity of the heptocyte is present t the cell surfce (24, 25) the lrge intrcellulr pool of ASGP receptors does not pper to supply the needed replcements. Our dt indicte tht the reutiliztion mechnism would hve to occur very rpidly to void trnsient decrese in the cell surfce receptor popultion. Nevertheless, the possibility remins tht such decrese in surfce receptors my hve occurred, but ws not detected in this experiment becuse reppernce of depleted receptors in the plsm membrne took plce during the 6-min incubtion of 2-5 C necessry for quntittion of receptor number. If this were true, it would men tht such recycling ws less sensitive to low temperture inhibition thn the process of lignd internliztion. Lysosomes hve been implicted in the ASGP receptor recycling pthwy by three observtions. First, ASGP binding ctivity hs been detected in lysosoml frctions from rt heptocytes, lthough the receptor ws pprently oriented with its binding site towrd the cytoplsm rther thn fcing the lysosoml lumen (22). Second, Tolleshug nd Berg (26) hve reported tht exposure of isolted heptocytes to chloroquine, drug tht inhibits degrdtion of proteins in lysosomes, results in decrese in the binding cpcity of heptocyte plsm membrnes to 15% of control levels. However, chloroquine reduced cell vibility, its effect ws not reversible, the WALL AND HUBBARD Receptor Distribution on Rt Heptocyte Surfces 695

10 concentrtion necessry to reduce binding cpcity ws higher thn the level needed to block lysosoml degrdtion, nd the postulted intrcellulr ccumultion of ASGP binding ctivity ws not substntited. Third, lignd-receptor dissocition in vitro occurs t low ph (<6) nd the lysosome is the only known orgnelle with such n cidic environment (27). Two of our present findings rgue ginst such suggestions of lysosoml involvement in the receptor slvge pthwy: () the inhibition of fusion of endocytic vesicles with lysosomes t 16 C hd no effect on the number of cell surfce receptors, nd (b) the lck of mesurble decrese in receptor number or chnge in their distribution in the temperture-shift experiments. Tken together, our observtions nd the results of others discussed bove indicte tht receptor reutiliztion occurs by very fst mechnism cpble of mintining constnt popultion of cell surfce receptors tht does not interchnge rpidly with much lrger intrcellulr pool. We would like to thnk Mr. H. Stukenbrok for invluble ssistnce with the electron microscopy, Ms. A. M for her technicl expertise, Ms. A. Shm nd Ms. M. Cmpo for preprtion of the mnuscript, nd Mr. T. Urquhrt ndms. P. Stenrd-Ossorio for theirphotogrphic work. This work ws supported by Ntionl Institutes of Helth grnt to A. L. Hubbrd (GM 29185) nd postdoctorl fellowship to D. A. Wll (GM 671). Received for publiction 23 Jnury 1981, nd in revisedform 27 April REFERENCES I. Neufeld, E, nd G. Ashwell Crbohydrte recognition systems for receptor-medited pinocytosis. In Biochemistry of Glycoproteins nd Proteoglycns.W. Lennrz, editor. Plenum Press, New York Wll, D. A, G. Wilson, nd A. L. Hubbrd The glctose-specific recognition system of mmmlin liver: the route of lignd internliztion in rt heptocytes. Cell. 21 : Hubbrd, A. L, nd H. Stukenbrok An electron microscope utordiogrphic study of the crbohydrte recognition systems in rt liver. II. Intrcellulr ftes of the -Ilignds. J. Cell Biol. 83: Dunn, W. A, J. H. LBdie, nd N.N. 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