Internalization of E. coli ST mediated by guanylyl cyclase C in T84 human colon carcinoma cells

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Biochimica et Biophysica Acta 1245 (1995) 29-36

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Internalization of E. coli ST mediated by guanylyl cyclase C in T84 human colon carcinoma cells R. Urbanski, S.L. Carrithers, S.A. Waldman * Department of Medicine and Pharmacology, Division of Clinical Pharmacology, Thomas Jefferson Universi~, 1100 Walnut Street, MOB 813, Philadelphia, PA 19107, USA Received 17 November 1994; accepted 24 March 1995

Abstract

Internalization of Escherichia coli heat-stable enterotoxin (ST) mediated by guanylyl cyclase C was examined in T84 human colon carcinoma cells. Surface-associated, receptor-bound ST was quantitatively separated from intracellular ligand employing acidic guanidineHC1. ST was internalized in a time-, temperature-, and ligand concentration-dependent fashion only by cells specifically expressing guanylyl cyclase C. Only receptors which bound reversibly to ST appeared to mediate endocytosis. The rate of internalization of ST empirically determined in these studies was 0.23 min- t. The density of surface receptors for ST was similar at 4°C and 37°C, suggesting that these receptors recycle back to the cell surface following internalization of ligand. Similarly, internalized ST was rapidly cleared from the intracellular compartment following endocytosis. These studies demonstrate that ST undergoes ligand-dependent receptor-mediated endocytosis in human colon carcinoma cells. Keywords: Guanylyl cyclase C; E. coli heat-stable enterotoxin; Internalization; T84 human colon carcinoma cell; ST receptor

1. Introduction

Enterotoxigenic Escherichia coli, an etiologic agent responsible for infectious diarrhea in developing countries, induces fluid and electrolyte secretion in the intestine by elaborating a low molecular weight heat-stable toxin, ST [1-5]. ST produced by E. coli is an 18 or 19 amino acid peptide that induces intestinal secretion and diarrhea upon binding to specific receptors, GCC, localized in the brush border of intestinal enterocytes [6-8]. These receptors are members of a family possessing ligand binding and guanylyl cyclase catalytic domains on a single transmembrane protein [8]. ST-receptor interaction leads to activation of guanylyl cyclase and increases in intracellular cGMP [6-8]. This cyclic nucleotide directly mediates fluid and electrolyte transport by activating a protein kinase and altering

Abbreviations: ANPs, atrial natriuretic peptides; CFTR, cystic fibrosis transmembrane regulator; DMEM, Dulbecco's minimal essential medium; cGMP, guanosine 3',5'-cyclic monophosphate; GCA, guanylyl cyclase A (ANP receptor); GCB, guanylyl cyclase B (C-type natriuretic peptide receptor); GCC, guanylyl cyclase C (heat-stable enterotoxin receptor); ST, heat-stable enterotoxin; PBS, phosphate-buffered saline * Corresponding author. Fax: + 1 (215) 9555681. 0304-4165/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 3 0 4 - 4 1 6 5 ( 9 5 ) 0 0 0 6 8 - 2

phosphorylation of, and chloride flux through, the cystic fibrosis transmembrane conductance regulator [9,10]. Endocytosis of ligand-receptor complexes is a common property of receptors which contributes to metabolism of the ligand, regulation of cell surface receptor density, and termination of the signaling cascade [l 1,12]. However, little is known concerning the role of endocytosis in the regulation of signaling mediated by receptor guanylyl cyclases. Natriuretic peptides are similar to ST in that they are low molecular weight heat-stable peptides which regulate cardiovascular homeostasis by interacting with specific guanylyl cyclase-associated receptors in various target tissues (GCA and GCB; [8]). Interestingly, there is heterogeneity in the ability of these different, but related, receptors to undergo receptor-mediated endocytosis. Thus, GCA receptors do not undergo internalization upon binding of ANP in cultured renal mesangial or renomedullary interstitial cells [13]. In contrast, GCB receptors are internalized upon binding of ligand and these receptors are actively recycled back to the cell surface following intracellular processing [ 14]. To date, the role of receptor-mediated endocytosis in transmembrane signaling induced by ST and the fate of this toxin bound to cell surface receptors remains undefined. The present study examines

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R. Urbanski et al. / Biochimica et Biophysica Acta 1245 (1995) 29-36

whether GCC undergoes internalization and the characteristics of that process in T84 cells derived from a human colonic adenocarcinoma metastatic to lung [15].

2. Materials and methods

quantify residual cell-associated [125I]ST, remaining cells were solubilized with 500 ~1 of 1 N NaOH for 60 min at 37°C. Dissociating agents tested included: acidic glycine buffer (50 mM glycine/150 mM NaC1, pH 2.5) acidic glycine buffer containing 2 M urea, 0.025% trypsin, and acidic guanidine buffer (2 M guanidine/150 mM NaCI, ph 2.5).

2.1. Materials T84 and COS-7 cells were obtained from the American Type Culture Company (Rockville, MD). ST was a generous gift of Dr. D. Robertson, Department of Biochemistry and Microbiology, University of Idaho, Moscow, Idaho. Tissue culture supplies were obtained from Gibco laboratories (Grand Island, NY). All other chemicals were of the highest analytical grade and obtained from Sigma (St. Louis, MO).

2.2. Cell culture T84 and COS-7 cells were cultured in l:l Dulbecco's modified Eagle's m e d i u m / H a m ' s F 12 (DMEM/F12) containing 5% ( v / v ) fetal bovine serum supplemented with penicillin and streptomycin at 37°C in an atmosphere of 5% CO2/95% 0 2 [16]. Media were replaced every 2 - 3 days. Cells were subcultured every 6 - 7 days. Cells were allowed to grow to confluence prior to subculturing into 24-well plates. After subculturing, confluent wells were incubated for an additional 24 h at 37°C prior to use in experiments. Studies were conducted with 10 6 cells/well as determined by manual counting using a hemocytometer. There was less than 10% variability in cell number/well.

2.3. lodination of ST ST was iodinated to a specific activity of 1000-2000 C i / m m o l and purified as described previously [16]. [125I]ST possesses full efficacy and potency in assays of receptor binding and guanylyl cyclase activity [ 16].

2.4. Dissociation of cell su~. ace-bound [1251]ST

2.5. Binding of [1251]ST to T84 cells in the absence of endocytosis Assays were performed at 4°C to quantify binding of [125I]ST to intact T84 cells in the absence of receptor endocytosis or recycling [ 11,12,14,18]. Confluent T84 cells in 24-well plates were washed twice with 500 /xl of binding media. Binding was initiated by the addition of 200 ~1 of binding media containing 10 nM [125I]ST. At various times binding was terminated by aspirating the media and washing the cells three times with ice-cold PBS to remove residual free [125I]ST. Cell surface-bound [JzsI]ST was recovered by incubating the cells with acidic guanidine buffer to dissociate [I25I]ST from its receptor, as described above. Residual cell-associated radioactivity, which represents endocytosed ligand, was recovered by solubilizing cells with 1 N NaOH for 1 h at 37°C. Nonspecific binding was determined in parallel incubations in the presence of a 100-fold excess of unlabeled ST.

2.6. Internalization of [125I]ST Internalization of [~25I]ST by T84 or COS-7 cells was quantified in assays performed at 37°C. Confluent cells in 24-well plates were washed twice with 500 /~1 of 37°C binding media prior to each experiment. Cells were incubated with 200 /zl of binding media containing increasing concentrations of [~25I]ST for various times up to 8 h, as indicated. At specific times, binding media was aspirated and cells washed three times with 500/zl of ice-cold PBS. Surface-bound and intracellular radioactivity was quantified as described above using acidic guanidine buffer and NaOH, respectively.

2.7. Miscellaneous Confluent T84 cells in 24-well plates were incubated with 10 nM [125I]ST in 200 /zl of buffer containing D M E M / H a m ' s F I 2, 0.1% bovine serum albumin, pH 7.4 (binding media) at 4°C for 3 h. After incubation, binding media was aspirated and cells washed three times with ice-cold PBS (500 /xl/wash) followed by addition of 500 /zl of ice-cold dissociating buffer and incubation for 10 min at 4°C. In some experiments, cells with [125I]ST bound to surface receptors were incubated with binding buffer containing 0.025% trypsin for 15 min at 4°C [17]. After incubation, dissociating buffer was aspirated and cells were washed with an additional 500 /zl of the same buffer. The two aliquots of dissociating buffer were combined and radioactivity quantified in a Packard gamma counter. To

Protein was determined as described by Bradford (BioRad, Richmond, CA). Binding isotherms for Scatchard analyses were plotted and binding constants calculated using 'Cigale' [16]. In some studies (FigsFig. 1.Fig. 2 Fig. 31, 3), double reciprocal plot analyses, in which the reciprocal of binding was plotted against the reciprocal of time, was employed to estimate equilibrium binding [19]. Linear regression analyses were performed using 'Cricket Graph' on a Macintosh IIci personal computer. In general, correlation coefficients for linear regression analyses were > 0.95. Results are representative of at least 2 experiments. Error bars represent standard error (S.E.) unless otherwise indicated.

R. Urbanski et al. / Biochimica et Biophysica Acta 1245 (1995) 29-36

31

100"

3. Results

3.1. Dissociation of [/251]ST from cell surface receptors Previous studies demonstrated that []25I]ST does not quantitatively dissociate from receptors [20-22]. Indeed, about 30% of specifically bound [125I]ST appears to be irreversibly associated with binding sites in intestinal mucosal cells [21]. Since quantification of internalized ST is predicated on the ability to completely dissociate and remove cell surface-bound ligand, agents demonstrated previously to dissociate ligand-receptor complexes were examined for their ability to dissociate [125I]ST from receptors on T84 cells. Of the dissociating agents tested, incubation with guanidine acidic buffer (2M guanidine/150 mM NaC1, pH 2.5) at 4°C for 10 min consistently removed greater than 95% of the surface-bound [125I]ST from T84 cells (Table 1). This is in close agreement with the ability of this agent to dissociate radiolabeled ST from receptors in cell-free assays [20]. Other agents were less effective or highly variable in their ability to quantitatively strip [ I:5I]ST from surface receptors of T84 cells. Interestingly, acidic glycine buffer is an effective stripping agent which quantitatively removes natriuretic peptides from guanylyl cyclase-coupled receptors, yet this buffer removed only about 80% of the [125I]ST from GCC on the surface of T84 cells [ 13,14]. Treatment of T84 cells with acidic guanidine buffer did not alter the integrity of the plasma membrane, since cells exposed to this buffer continued to exclude trypan blue.

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Fig. 2. Time-course of cell surface binding and internalization of increasing concentrations of [tESI]ST in T84 cells at 37°C. Incubations were conducted and non-specific binding was quantified as described above. Internalized ligand was separated from cell surface-bound ligand with dissociation buffer as described above. Concentrations of labeled ST employed included 0.1 nM (triangles), I nM (open squares), and 10 nM (circles). Upper panel, total cell-associated ligand; middle panel, cell surface-bound ligand; lower panel, internalized ligand. Closed squares represent the total cell-associated, surface-bound, and internalized radioactivity when COS-7 cells, which do not possess GCC, were incubated with l0 nM [J25I]ST. These studies are representative of at least three experiments.

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Fig. 1. Binding and internalization of [=25I]ST to T84 cells at 4°C. T84 cells (106 cells/well) were incubated with 10 -8 M [t25I]ST and incubated for 2.5 h to equilibrium, as described in Section 2. Specific binding was calculated by subtracting non-specific binding, determined in parallel incubations in the presence of e x c e s s ( 1 0 - 6 M ) unlabeled ST, from total binding. At equilibrium, cells containing total cell-associated radioactivity (circles) were washed with dissociation buffer to remove cell surfacebound-radioactivity (squares). The radioactivity remaining with these washed cells represents radioactivity in the intracellular compartment (internalized; triangles). Results are representative of at least three experiments. Error bars, S.E.

[125I]ST bound to T84 cells at 4°C in a time-dependent and saturable fashion (Fig. l). Employing 10 -8 M ligand, binding equilibrium was achieved in about 60 min. Analysis of these data by double reciprocal plot demonstrated that maximum binding of [I25I]ST was 50 fmole ST//106 cells, using 10 -8 M ligand. There was no internalization of [JzSI]ST in T84 cells at 4°C, since virtually all of the radioactivity associated with cells could be dissociated using acidic guanidine buffer. These data agreed closely with earlier studies demonstrating minimal internalization of ligands by receptor-mediated endocytosis at 4°C [11,12,17]. In agreement with these observations, cell surface ST receptor density remained constant throughout the

32

R. Urbanski et al. / Biochimica et Biophysica Acta 1245 (1995) 29-36 3.4. Internalization o f [1251]ST at 37°C

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3 h incubation in the presence of [125I]ST, once equilibrium was achieved. These observations demonstrate that internalization of [125I]ST does not occur and there is no appreciable down-regulation of ST receptors at 4°C despite persistent exposure to free ligand. 3.3. Binding o f [125I]ST to T84 cells at 37°C Total radioactivity reflecting the association of [ J25I]ST with T84 cells increased in a time-and concentration-dependent fashion upon incubation of these cells with increasing concentrations of that ligand at 37°C (Fig. 2A). Association of radiolabeled ligand with T84 cells was specific since that association could be completely blocked by incubation with an excess of unlabeled ST. Furthermore, cell-associated radioactivity could not be detected when the highest concentration of [12SI]ST (10 -8 M) was incubated with COS-7 cells, which do not express G C C receptors, for 8 h [23]. Total radioactivity associated with T84 cells resulting from their incubation with [~25I]ST at 37°C could reflect ligand bound to cell surface receptors a n d / o r radioactivity in the intracellular compartment reflecting internalized liga n d - r e c e p t o r complexes. ST associated with surface receptors was quantified at various times by incubating T84 cells with acidic guanidine-HC1, as described above (Fig. 2B). ST bound to cell surface receptors in a concentrationand time-dependent and saturable fashion. Cell surface binding appeared to reach equilibrium at 37°C in a concentration-independent fashion, with equilibrium achieved by 60 min with all ligand concentrations examined (data not shown). [125I]ST binding to the surface of T84 cells is specific since it can be completely competed with excess unlabeled ligand. Similarly, COS-7 cells do not exhibit measurable binding of this ligand at the highest concentrations studied (10 -8 M).

[125I]ST internalized by endocytosis was quantified at each time point by dissolving cells, whose surface receptor-associated ligand previously had been dissociated with acidic guanidine buffer, with 1,0 N NaOH, as described above (Fig. 2C). Radioactivity increased in T84 cells incubated with increasing concentrations of [L25I]ST at 37°C in a time-and concentration-dependent fashion. This is in contrast to results obtained at 4°C, in which radioactivity could not be detected in the intracellular compartment after dissociating ligand from surface receptors with acidic guanidine (Fig. 1). Thus, accumulation of ST in the intracellular compartment is a temperature-dependent process. Also, accumulation of ST intracellularly could be completely competed by performing these experiments in the presence of excess unlabeled ligand. These data suggest that intracellular accumulation of ST is mediated by specific receptors. In close agreement with these observations, radioactivity could not be detected in the intracellular compartment of COS-7 cells, which lack ST receptors, when these cells were incubated with 10 -8 M [~25I]ST. The specificity, temperature sensitivity, and concentrationand time-dependence of the accumulation of radioactivity in T84 cells suggests that ST is internalized by a receptormediated process involving the specific interaction of the ligand and GCC. 3.5. Down-regulation and receptor recycling o f GCC The density of surface receptors for ST appear to be similar on T84 cells at 4°C and 37°C (compare Fig. 1 and Fig. 2B). These data suggest that the steady-state concen-

Table 1 The effect of various dissociating agents on the binding of [125I]ST to T84 cells at 4°C Treatment ~ % [125I]STdissociated from cell surface receptors b None 0.2 M acetic acid/0.5 M NaC1, pH 2.5

0 60+6

50 mM glycine/0.1 mM NaC1, pH 2.5

83+9

2 M guanidine-HCl/0.15 M NaCI, pH 2.5

98 _+1

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35 + 1

a T84 cells were incubated with 10 nM [125I]ST at 4°C for 2 h, to equilibrium, and subsequently incubated with the dissociating buffer as described in Section 2. Studies were conducted at 4°C to minimize endocytosis of ligand-receptor complexes. b % [ 125I]STdissociated from cell surface receptors = (radioactivity associated with cells after incubation with dissociating agent)/(radioactivity associated with cells before incubation with dissociating agent). Dissociation of [I251]ST from cells with trypsin was performed as described in Section 2.

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R. Urbanski et al. / Biochimica et Biophysica Acta 1245 (1995) 29-36

tration of GCC at the surface of these cells is temperatureand ligand-independent. To examine this further, T84 cells were incubated with near-saturating concentrations (10 nM) of [~25I]ST at 4°C and 37°C for up to 3 h and specific cell surface-bound ligand was quantified at various times (Fig. 3). Binding to cell surface receptors was time-dependent and saturable at both temperatures. Equilibrium was achieved rapidly, at the earliest time point sampled (15 min), at 37°C whereas it was achieved more slowly (45 min) at 4°C, as expected. Maximum binding achieved at equilibrium at 10 -8 M, calculated by double reciprocal plot analysis of the time course of binding of [~25I]ST to cell surface receptors, was similar at 37°C (50 + 3 fmol/106 cells) and 4°C (55 + 7 fmol/106 cells). As demonstrated above, there is no detectable internalization of [125I]ST at 4°C. Therefore, receptor density quantified at this temperature represents the full complement of GCC at the cell surface, in the absence of endocytosis. The number of surface receptors for ST on T84 cells were similar at 4°C and 37°C, demonstrating that GCC does not undergo down-regulation, desensitization, or depletion from the cell surface upon persistent exposure to ligand. Similarly, that receptor density is unchanged at 4°C and 37°C, although internalization of ligand occurs at the higher temperature, suggests that GCC may be rapidly recycled back to the

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Fig. 5. Kinetics of internalization of [m251]ST bound at 4°C to T84 cells which were subsequently increased to 37°C. Studies were performed as outlined in Fig. 4 and radioactivity in the intracellular compartment was quantified as described in Section 2. Inset, semilogarithmic plot of the time course of radiolabeled ST bound to the cell surface (squares) or free in the media (circles; from Fig. 4). These studies are representative of at least three experiments.

membrane following ligand-induced endocytosis. Rapid recycling of cell surface receptors, without down-regulation at the cell surface, following receptor endocytosis has been observed previously [11,12,17,26-32].

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Fig. 4. Kinetics of [m25I]ST bound at 4°C to T84 cells which were subsequently increased to 37°C. T84 cells (106 cells/well) were incubated with 10 nM [m25I]ST for 2.5 h to equilibrium at 4°C. At the end of incubation, free radioligand was removed, cells were washed three times with ice-cold binding buffer, and washed cells were placed in binding buffer warmed to 37°C. At various times, radioactivity free in the media (circles) and bound to the surface of cells (squares) was quantified as described in Section 2 (A). The time-course of the association of radioactivity with the cell surface after transfer of cells to 37°C was analyzed on a semilogarithmic plot (B). These studies are representative of at least three experiments.

The fate of [125I]ST bound to surface receptors in T84 cells was directly examined. In these experiments, cell surface ST receptors were pre-bound with [125I]ST by incubating T84 cells with 10 nM [I25I]ST at 4°C for 2.5 h. At this temperature, there is no internalization of radioligand and binding reaches equilibrium within 45 min (see Fig. 1,3). After incubation at 4°C, cells were placed at 37°C with fresh binding media which did not contain free [~zSI]ST and the fate of cell surface-bound radioactivity was followed (FigsFig. 4.Fig. 5 4, 5). When cells were warmed from 4°C to 37°C, there was a rapid decrease in cell-associated [125I]ST within the first 9 min, to about 30% of that initially bound at 4°C (Fig. 4A). A parallel rapid increase in radioligand was observed in the binding media. The semilogarithmic plot of the loss of cell-associated radioactivity exhibits a curvilinear isotherm, suggesting multiple processes a n d / o r receptor populations contributing to different rates of loss of ST from cells (Fig. 4B). The initial rapid rate of loss of cell-associated radioactivity most likely corresponds to both dissociation of ligand-receptor complexes at the surface and degredation or recycling and release of ST at the cell surface. The terminal ( > 15 rain) rate of loss of cell-associated radioactivity is very slow and occurs in the absence of internaliza-

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R. Urbanski et al. / Biochimica et Biophysica Acta 1245 (1995) 29-36

tion (see below and Fig. 5). This slow rate of loss of radioactivity may reflect binding sites characterized previously as non-dissociable [20,21]. They represent about 25% of the total initial radioactivity associated with the cell surface, consistent with previous observations [20,21]. The mechanisms underlying slow dissociation of ST-receptor complexes and their functional significance remain unclear. 3.7. Kinetics of [125I]ST internalization in T84 cells [ 12sI]ST increased in the intracellular compartment in a time-dependent fashion (Fig. 5). Internalized radioactivity increased over an interval during which rapid loss of radioactivity from cells occurred (0 to 12 min). However, internalization appeared to be biphasic and the quantity of radioactivity internalized decreased to nearly undetectable levels after 12 min. The time-course of this decrease in internalization precisely corresponded to that of the decrease in dissociable, and the increase in non-dissociable, binding sites at the cell surface (see Fig. 4). Indeed, intracellular radioactivity decreased to a minimum when only non-dissociable binding sites remained at the cell surface. These data suggest that non-dissociable ST binding sites do not contribute to endocytosis of that ligand in T84 cells. In addition, the decrease in radioactivity in the intracellular compartment over time demonstrates that ST entering the cell by receptor-mediated endocytosis is also removed from the cell. The mechanisms by which cells clear ST from the intracellular compartment, including retroendocytosis a n d / o r metabolism of internalized ligand, is the focus of current studies in this laboratory. In order to estimate the rate of internalization of ST, the rates of loss of cell surface radioactivity and accumulation of radioactivity in the media were compared (Fig. 5, inset). The rate of loss of cell surface radioactivity reflects dissociation of ligand-receptor complexes into the media and endocytosis of those complexes. Therefore, ksfc = kint + kdi ....

where ksf c is the rate constant of loss of radioactivity from dissociable sites at the cell surface, kin t is the rate constant of internalization, and kdissoc is the rate constant of dissociation of ligand-receptor complexes. The rate constant of loss of cell surface radioactivity from dissociable sites can be quantified from the semilogarithmic plot of the data presented in Fig. 4, correcting the total number of binding sites for only dissociable sites (76%; Fig. 5, inset). The relative quantity of dissociable and non-dissociable sites was estimated from the Y-intercepts of the semilogarithmic plot of the time course of loss of cell surface radioactivity (Fig. 4B). The rate constant of dissociation of ligand-receptor complexes can be directly estimated from the semilogarithmic plot of the accumulation of radioactivity in the media (Fig. 5, inset). The difference between these rate constants represents an estimate of the contribution of

the rate of internalization to the rate of loss of radioactivity from the cell surface. The rate constants of loss of radioactivity from the cell surface and of dissociation, estimated from the slopes of the semilogarithmic plots, were k~fc = 0.42 + 0.08 m i n - l ; kdi.... = 0.14 + 0.01 min - l , respectively. Employing these values, the calculated rate of internalization of GCC w a s : kin t = 0.28 min- ~. This rate of internalization is rapid and comparable to that of other ligand-receptor complexes which undergo ligand-induced receptor-mediated endocytosis, including growth factor receptors [33].

4. Discussion

The present studies demonstrate that ST in human colonic tumor cells in vitro undergo ligand-induced receptor-mediated endocytosis. Endocytosis of ST is time-, ligand concentration-, and temperature-dependent. Althoughendocytosis is temperature-dependent, receptor concentra-tion at the cell surface is similar at 4°C and 37°C, suggesting that cell surface GCC molecules do not undergo desensitization or down-regulation and are rapidly replenished after internalization. Whether the cell surface complement of GCC is maintained by rapid recycling of receptors, recruitment from a cytoplasmic pool of these receptors, or new protein synthesis remains to be elucidated [11,12,17,30,31]. There are at least 2 functional types of ST binding sites in these cells: those which undergo dissociation from ligand and those which appear to be non-dissociable [20,21]. The present data suggest that it is only the dissociable sites that undergo endocytosis. Endocytosis of GCC appears to be a rapid event, comparable to that of other peptide receptors, including growth factor receptors. Once ligand-receptor complexes have been internalized, ST is cleared from the cell. Whether clearance of ligand from intracellular sites occurs by metabolism or retroendocytosis has not been defined. These studies are the first to demonstrate and define the characteristics of endocytosis of ST mediated by GCC. Previous studies have assessed radiolabeled ST binding to T84 cells at 4°C and 37°C [15]. In these studies, the quantity of surface-bound ST obtained at 4°C was observed to be about 70% of that noted at 37°C. These differences are consistent with the present observations that at 4°C the amount of radioactivity associated with T84 cells reflects ligand bound to surface receptors only whereas that at 37°C reflects ligand bound to surface receptors and internalized. Indeed, the differences between the amount of radioactivity associated with T84 cells at different temperatures observed in the earlier studies are comparable to those reported herein. The authors of those earlier studies suggested that the differences in radioactivity associated with T84 cells at different temperatures may reflect endocytosis of ligand-receptor complexes [15].

R. Urbanski et al. / Biochimica et Biophysica Acta 1245 (1995) 29-36

Endocytosis of peptide receptor-coupled guanylyl cyclases has been examined previously and appears to be heterogeneous for this family of proteins. In cultured mesangial and renomedullary interstitial cells, ANP-GCA interaction did not mediate internalization of liganded receptors [13]. Rather, signal termination in this system appeared to be mediated by a very rapid dissociation of ANP from receptors. This rapid dissociation of ligand from GCA is similar to the observations in the present study. Thus, the dissociation rate constant of radiolabeled ST from intact T84 cells, 0.13 min -~, is much greater than that rate constant determined previously for these receptors in cell-free systems, 0.016 min- 1 [21 ]. These observations suggest that rapid dissociation of ligand-receptor complexes may play a prominent role in signal termination by guanylyl cyclase-coupled receptors [13]. The precise mechanisms underlying acceleration of the dissociation rate of these receptors in intact cells compared to cell-free membranes remain to be elucidated. In contrast to GCA in renal cells, GCB undergoes ligand-dependent internalization in PC 12 cells in vitro [ 14]. In these studies, endocytosis of GCB is accompanied by a rapid down-regulation of cell surface receptors. These observations contrast with the results reported herein, that internalization is not accompanied by down-regulation of GCC in T84 cells. However, it is notable that in the earlier studies, down-regulation was quantified by incubating cells with high concentrations of unlabeled ANP to saturate receptors, removing excess unlabeled ligand, and subsequently measuring cell surface receptors remaining by incubation with labeled ligand. In these studies, if the dissociation rate of ligand-receptor complexes is not sufficiently rapid to permit occupancy by labeled ligand over the time-course of the second incubation, then saturated receptors will remain bound to unlabeled ligand and will be undetectable [ 17]. Thus, the contribution of down-regulation to endocytosis of GCB receptors remain unclear. In addition, these studies suggested that GCB recycles back to the cell surface from an intracellular pool of receptors subsequent to internalization [14]. Therefore, rapid recycling may be the mechanism by which T84 cells maintain their complement of ST receptors during internalization. This mechanism is similar to those operating in other peptide ligand-receptor coupled systems including ANP clearance and epidermal growth factor receptors, which are recycled back to the cell surface from internal pools [17,30,31,34]. Ligand-induced receptor-mediated internalization is first-order with respect to the concentration of the ligandreceptor complex at the cell surface [18,35]. Typically, the rate of ligand-induced receptor internalization can be estimated directly in experiments in which cell surface receptors are preloaded in the absence of internalization at 4°C and then transferred to 37°C to initiate internalization [33,36]. However, these studies are predicated on the rate of dissociation of ligand-receptor complexes being much

35

lower than the rate of internalization, in order for the change in concentration of cell surface receptor-ligand complexes to reflect internalization only [18,35]. In the present studies, a direct determination of internalization rate was not possible because the dissociation rate of ligand-receptor complexes approached that of internalization. Despite this rapid dissociation of cell surface-bound ST, the rate of internalization could be estimated, under these incubation conditions, by calculating the difference between the loss of cell surface and cell-associated radioactivity. It should be noted that this estimate of the rate of internalization of GCC represents a lower limit. That is, internalized radioactivity is rapidly cleared from the cell into the extracellular media (see Fig. 5). Radioactivity cleared from the intracellular to the extracellular compartment results in a potential overestimation of the rate of dissociation of ligand-receptor complexes and, consequently, an underestimation of the rate of internalization. Although the estimate for the rate of internalization of GCC presented herein is only a lower limit, it demonstrates that these peptide receptors undergo rapid internalization in T84 cells. Rapid rates of internalization of peptide ligands have been demonstrated previously in other systems including insulin, EGF, and angiotensin II [33]. Also, the time-course of internalization of radiolabeled ST in T84 cells described herein is almost identical to that of radiolabeled ANP in rat vascular smooth muscle cells possessing guanylyl cyclase-coupled receptors [37]. Finally, rapid internalization and repletion of cell surface constituents, with a time-course similar to that described herein for ST receptors, has been described recently for the CFFR [30]. It is especially noteworthy that these studies were conducted in T84 cells, that the CFTR is located in the same subcellular compartment as GCC, and that the CFTR is the ultimate effector for the product of GCC, cGMP [9,10]. Studies presented herein suggest a model for internalization of GCC. Endocytosis is initiated by association of ST with the extracellular ligand binding domain of GCC. Ligand-receptor interaction may induce alterations in GCC that result in clustering of these receptors in coated pits. Coated pit-mediated endocytosis of GCC is consistent with the observation that this receptor generates a second messenger, cGMP, upon ligand binding [ 11,12,17,24]. Furthermore, ligand-induced receptor-mediated endocytosis depends on specific interactions between cytoplasmic domains of receptors and components of the endocytic apparatus [ 11,12,25,38,39]. Recent studies suggest that specific consensus sequences, YXXZ and LZ (where Z indicates one of the following hydrophobic amino acids: L, I, V, M, C, A) in the cytoplasmic domain of receptors are required for ligand-induced, coated pit-mediated receptor endocytosis [38,39]. Indeed, receptors which undergo ligandinduced receptor mediated endocytosis, such as those for epidermal growth factor, insulin-like growth factor II, and vesicular stomatitis virus glycoprotein G, possess in their

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R. Urbanski et al. / Biochimica et Biophysica Acta 1245 (1995) 29-36

cytoplasmic domains the consensus sequence YXXZ [3942]. Of significance, this sequence also appears in the carboxy-terminal region of GCC, supporting the suggestion that endocytosis of this receptor is mediated by mechanisms involving coated pits. Once GCC is incorporated into coated pits, ligand-receptor complexes may be routed to the early endosomal compartment [30,43-47]. In this compartment there is rapid sorting of ligand-receptor complexes with routing of some portion of these receptors back to the cell surface [30,43-47]. The quantity of receptors in the intracellular pool which is rapidly recycling back to the cell surface remains unclear. Similarly, whether ST dissociates from internalized receptors permitting recycling of unliganded receptors, or whether ST undergoes retroendocytosis is unknown. Finally, it is unclear whether a portion of the internalized ST or GCC is routed from the early endosomal compartment to lysosomes for degradation. These questions are currently being examined in this laboratory.

Acknowledgements This research was supported, in part, by grants from the W.W. Smith Charitable Trust, the National Institutes of Health (1 R55 DK43805), the National Science Foundation (IBN-9205717) and the Elsa U. Pardee Foundation. Ray Urbanksi was the recipient of a Pharmaceutical Manufacturers Association Postdoctoral Fellowship in Clinical Pharmacology. Stephen L. Carrithers was the recipient of an NIH Postdoctoral Fellowship (1 F32 CA63764-01).

References [1] Ryder, R.N., Wachsmuth, I.K., Buxton, A.E, Evans, D.G., DuPont, H.L., Mason, E. and Barrett, F.F. (1976) N. Engl. J. Med. 295, 849-853. [2] Evans, D.G., Olarte, J., DuPont, H.L., Evans, D.J., Galinda, E., Portnoy, B.L. and Conklin, R.H. (1977) J. Ped. 91, 65-68. [3] Rohde, J.E. (1984) Rev. Infect. Dis. 6, 840-854. [4] Gold, R. (1988) Drugs 36 (Suppl. 4), 1-5. [5] Echeverria, P., Taylor, D.N., Lexsomboon, U., Bhaibulaya, M., Blacklow, N.R., Tamura, K. and Sakazaki, R. (1989) J. Infect. Dis. 159, 543-548. [6] Thompson, M.R. (1987) Immunopathol. Res. 6, 103-116. [7] Hugues, M. and Waldman, S.A. (1992) Biochem. (Life Sci. Adv.) 10, 103-112. [8] Fulle, H.-J. and Garbers, D.L. (1994) Cell Biochem. Func. 12, 157-165. [9] Tien, X.-Y., Brasitus, T.A., Kaetzel, M.A., Dedman, J.R. and Nelson, D.J. (1994)J. Biol. Chem. 269, 51-54. [10] Chao, A.C., De Sauvage, F,J., Dong, Y.J., Wagner, J.A., Goeddel, D.V, and Gardner, P. (1994) EMBO J. 13, 1065-1072. [11] Wileman, T., Harding, C. and Sahl, P. (1985) Biochem. J. 232, 1-14. [12] Trowbridge, I.S., Collawn, J.F. and Hopkins, C.R. (1993) Annu. Rev. Cell Biol. 9, 129-161.

[13] Koh, G.Y., Nussenzveig, D.R., Okolicany J., Price, D.A. and Maack, T. (1992)J. Biol. Chem. 267, 11987-11994. [14] Rathinavelu, A. and Isom, G.E. (1991) Biochem. J. 276, 493-497. [15] Guarino, A., Cohen, M., Thompson, M., Dharmsathaphorn, K. and Giannella, R. (1987) Am. J. Physiol. 253, G775-G780. [16] Carrithers, S.L., Parkinson, S.J., Goldstein, S., Park, P., Robertson, D.C. and Waldman, S.A. (1994) Gastroenterology 107, 1653-1661. [17] Nussenzweig, D.R., Lewicki, J.A. and Maack, T. (1990) J. Biol. Chem. 265, 20952-20958. [18] Owensby, D.A., Morton, P.A. and Schwartz, A.L. (198) Methods Cell Biol. 32, 305-328. [19] Waldman, S.A., Phillips, K. and Parkinson, S.J. (1994) J. Infect. Dis. 170, 1498-1507. [20] Dreyfus, L.A. and Robertson, D.C. (1984) Infect. Immun. 46, 537-543. [21] Hugues, M., Crane, M.R., O'Hanley, P. and Waldman, S.A. (1991) Biochemistry 30, 10738-10745. [22] Katwa, L.C., Parker, C.D., Dybing, J.K. and White, A.A. (1993) Arch. Biochem. Biophys. 304, 338-344. [23] Schulz, S., Green, C.K., Yuen, P.S.T. and Garbers, D.L. (1990) Cell 63, 941-948. [24] Sharma, R.J., Woods, N.M., Cobbold, P.H. and Grant, D.A.W. (1989) Biochem. J. 259, 81-89. [25] Lamaze, C., Baba, T., Redelmeir, T.E. and Schmid, S.L. (1993) Mol. Biol. Cell 4, 715-727. [26] Hopkins, C.R. and Trowbridge, I.S. (1983) J. Cell Biol. 97,508-521. [27] Krupp, M.N., Connolly, D.T. and Lane, M.D. (1982) J. Biol. Chem. 257, 11489-11496. [28] Stotscheck, C.M. and Carpenter, G. (1984) J. Cell Physiol. 120, 296-302. [29] Stoscheck, C.M. and Carpenter, G. (1984) J. Cell Biol. 98, 10481053. [30] Sorkin, A., Krolenko, S., Kudrjavtceva, N., Lazebnik, J., Teslenko, L., Soderquist, A.M. and Nikolsky, N. (1991) J. Cell Biol. 112, 55-63. [31] Prince, L.S., Workmann, R.B., Jr. and Marchase, R.B. (1994) Proc. Natl. Acad. Sci. USA 91, 5192-5196. [32] Huecksteadt, T., Olefsky, J.M., Bradenberg, D. and Heidenreich, K.A. (1986) J. Biol. Chem. 261, 8655-8659. [33] Waters, C.M., Oberg, K.C., Carpenter, G. and Overholser, K.A. (1990) Biochemistry 29, 3563-3569. [34] Gladhaugh, I.P. and Christofferson, T. (1988) J. Biol. Chem. 263, 12199-12203. [35] Schwartz, A.L., Fridovich, S.E. and Lodish, H.F. (1982) J. Biol. Chem. 257, 4230-4237. [36] Pandey, K.N. (1992) Biochem. J. 288, 55-61. [37] Cahill, P.A., Redmond, E.M. and Keenan, A.K. (1990) J. Biol, Chem. 265, 21896-906 [38] Sandoval, I.V. and Bakke, O. (1994) Trends Cell Biol. 4, 292-297, [39] Johnson, K.F., Chart, W. and Kornfeld, S. (1990) Proc. Natl. Acad. Sci. USA 87, 10010-10014. [40] Canfield, W.M., Johnson, K.F., Ye, R.D., Gregory, W. and Kornfeld, S. (1991) J. Biol. Chem. 266, 5682-5688. [41] Thomas, D.C. and Roth, M.G. (1994) J. Biol. Chem. 269, 1573215739. [42] French, A.R., Sudlow, G.P., Wiley, H.S. and Lauffenburger, D.A. (1994) J. Biol. Chem. 269, 15749-15755. [43] Hubbard, A.L., Wall, D.A. and Ma, A. (1983) J. Cell Biol. 61, 188-200.

[44] Geuze, H.J., Slot, J.W. and Strous, G.J.A.M. (1983) Cell 32, 277287. [45] Miller, K., Beardmoe, J., Kanety, H., Schlessinger, J. and Hopkins, C., R. (1986) J. Cell Biol. 102, 500-509. [46] Geuze, H.J., Slot, J.W. and Schwartz, A.L. (1987) J. Cell Biol. 104, 1715-1723. [47] Renfrew, C.A. and Hubbard, A.L. (1991) J. Biol. Chem. 266, 4348-4356.