MYLAN EXHIBIT - 1065
`Mylan Pharmaceuticals, Inc. v. Bausch Health Ireland, Ltd.
`IPR2022-00722
`
`

`

`30
`
`R. Urbanski et al. / Biochimica et Biophysica Acta 1245 (1995) 29-36
`
`whether GCC undergoesinternalization and the character-
`istics of that process in T84 cells derived from a human
`colonic adenocarcinoma metastatic to lung [15].
`
`2. Materials and methods
`
`2.1. Materials
`
`T84 and COS-7 cells were obtained from the American
`Type Culture Company (Rockville, MD). ST was a gener-
`ous gift of Dr. D, Robertson, Department of Biochemistry
`and Microbiology, University of Idaho, Moscow, Idaho.
`Tissue culture supplies were obtained from Gibco laborato-
`ries (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 1:1 Dulbecco’s
`modified Eagle’s medium/Ham’s F12 (DMEM/F12) con-
`taining 5% (v/v) fetal bovine serum supplemented with
`penicillin and streptomycin at 37°C in an atmosphere of
`5% CO,/95% O, [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° cells/well
`as determined by manual counting using a hemocytometer.
`There was less than 10% variability in ce!l number/well.
`
`2.3. lodination of ST
`
`ST was iodinated to a specific activity of 1000-2000
`Ci/mmol and purified as described previously [16].
`['*°1]ST possesses full efficacy and potency in assays of
`receptor binding and guanylyl cyclaseactivity [16].
`
`2.4. Dissociation of cell surface-bound ['?°1]ST
`
`Confluent T84 cells in 24-well plates were incubated
`with 10 nM ['*I]ST in 200 yl of buffer containing
`DMEM/Ham’s F12, 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 y1/wash) followed by addition of 500
`1 of ice-cold dissociating buffer and incubation for 10
`min at 4°C. In some experiments,cells with ['*°I]JST 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 jz] of the same buffer.
`The two aliquots of dissociating buffer were combined and
`radioactivity quantified in a Packard gamma counter. To
`
`quantify residual cell-associated ['*°I]ST, remaining cells
`were solubilized with 500 wl of 1 N NaOH for 60 min at
`37°C. Dissociating agents tested included: acidic glycine
`buffer (SO mM glycine/150 mM NaCl, pH 2.5) acidic
`glycine buffer containing 2 M urea, 0.025% trypsin, and
`acidic guanidine buffer (2 M guanidine /150 mM NaCl, ph
`2.5).
`
`2.5. Binding of ['?HST to T84 cells in the absence of
`endocytosis
`
`Assays were performed at 4°C to quantify binding of
`[51]ST to intact T84 cells in the absence of receptor
`endocytosis or recycling [11,12,14,18]. Confluent T84cells
`in 24-well plates were washed twice with 500 wl of
`binding media. Binding was initiated by the addition of
`200 yl of binding media containing 10 nM ['?°I]ST. At
`various times binding was terminated by aspirating the
`media and washingthe cells three times with ice-cold PBS
`to remove residual
`free [!*°IJST. Cell
`surface-bound
`['°SI]JST was recovered by incubating the cells with acidic
`guanidine buffer to dissociate ['**IJST 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. Non-
`specific binding was determined in parallel incubations in
`the presence of a 100-fold excess of unlabeled ST.
`
`2.6. Internalizationof [!°°1]ST
`
`Internalization of ['>1]JST 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 yl of 37°C
`binding media prior to each experiment. Cells were incu-
`bated with 200 wl of binding media containing increasing
`concentrations of ['?SIJST for various times up to 8 h, as
`indicated. At specific times, binding media was aspirated
`and cells washed three times with 500 x1 of ice-cold PBS.
`Surface-bound and intracellular radioactivity was quanti-
`fied as described above using acidic guanidine buffer and
`NaOH,respectively.
`
`2.7. Miscellaneous
`
`Protein was determined as described by Bradford (Bio-
`Rad, 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 recip-
`rocal of binding was plotted against the reciprocal of time,
`was employedto estimate equilibrium binding [19]. Linear
`regression analyses were performed using ‘Cricket Graph’
`on a Macintosh IIci personal computer. In general, correla-
`tion 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 indi-
`cated.
`
`

`

`R. Urbanski et al. / Biochimica et Biophysica Acta 1245 (1995) 29-36
`
`31
`
`100 ~wi
`
`Oo
`
`2
`
`6
`4
`Time (hours)
`
`8
`
`10
`
`3. Results
`
`3.1. Dissociation of ['?>1]STfrom cell surface receptors
`
`Previous studies demonstrated that [!?°IJST does not
`quantitatively dissociate from receptors [20-22]. Indeed,
`about 30% of specifically bound {'°IJST appears to be
`irreversibly associated with binding sites in intestinal mu-
`cosal 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 {'*°IJST from re-
`ceptors on T84 cells. Of the dissociating agents tested,
`incubation with guanidine acidic buffer (2M guanidine/ 150
`mM NaCl, pH 2.5) at 4°C for 10 min consistently removed
`greater than 95% of the surface-bound ['?°I]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
`['°1]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 ['*IJST from GCConthesurface 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.
`
`FmolesofSTCell-Associated/Well
`Total
`FmoalesofSTSurface-Bound/Well
`FmolesofSTInternalized/Well
`
`FmolesofSTBound/Well
`
`0
`
`200
`
`100
`Time (min)
`Fig. 1. Binding and internalization of ['**IJST to T84 cells at 4°C, T84
`cells (10° cells/well) were incubated with 10-8 M ['*5I]ST and incu-
`bated for 2.5 h to equilibrium, as described in Section 2. Specific binding
`was calculated by subtracting non-specific binding, determinedin parallel
`incubations in the presence of excess (10° M)unlabeled ST, from total
`binding. At equilibrium,cells containing total cell-associated radioactivity
`(circles) were washed with dissociation buffer to remove cell surface-
`bound-radioactivity (squares). The radioactivity remaining with these
`washed cells represents radioactivity in the intracellular compartment
`(internalized; triangles). Results are representative of at least three experi-
`ments. Error bars, S.E.
`
`Fig. 2. Time-course of cell surface binding and internalization of increas-
`ing concentrations of ['**IJST 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),
`1 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 ra-
`dioactivity when COS-7 cells, which do not possess GCC, were incubated
`with 10 nM ['SI]ST. These studies are representative of at least three
`experiments.
`
`3.2. [!?°I]ST binding to T84 cells at 4°C
`
`['51]ST bound to T84 cells at 4°C in a time-dependent
`and saturable fashion (Fig. 1). Employing 10~® M ligand,
`binding equilibrium was achieved in about 60 min. Analy-
`sis of these data by double reciprocal plot demonstrated
`that maximum binding of['*5I]ST was 50 fmole ST/10°
`cells, using 10~* M ligand. There was nointernalization
`of [’SI]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 sur-
`face ST receptor density remained constant throughout the
`
`

`

`32
`
`R. Urbanski et al. / Biochimica et Biophysica Acta 1245 (1995) 29-36
`
`Surface/Well
`FmolesofSTBoundtotheCell
`
`3.4, Internalization of [’?°UST at 37°C
`
`['*SJST internalized by endocytosis was quantified at
`each time point by dissolving cells, whose surface recep-
`tor-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 ['?°I]ST at
`37°C in a time-and concentration-dependent fashion. This
`is in contrast to results obtained at 4°C, in which radioac-
`tivity could not be detected in the intracellular compart-
`ment after dissociating ligand from surface receptors with
`acidic guanidine (Fig. 1). Thus, accumulation of ST in the
`intracellular compartment is a temperature-dependent pro-
`cess. Also, accumulation of ST intracellularly could be
`completely competed by performing these experiments in
`the presence of excess unlabeled ligand. These data sug-
`gest that intracellular accumulation of ST is mediated by
`specific receptors. In close agreement with these observa-
`tions,
`radioactivity could not be detected in the intra-
`cellular compartment of COS-7 cells, which lack ST recep-
`tors, when these cells were incubated with 107° M ['?51]ST.
`The specificity, temperature sensitivity, and concentration-
`and time-dependence of the accumulation of radioactivity
`in T84 cells suggests that ST is internalized by a receptor-
`mediated process involving the specific interaction of the
`ligand and GCC.
`
`3.5, Down-regulation and receptor recycling of 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 |
`The effect of various dissociating agents on the binding of ['**I]ST to
`T84 cells at 4°C
`
`Treatment *
`
`None
`
`% [']|ST dissociated
`from cel! surface
`receptors °
`0
`
`0.2 M acetic acid /0.5 M NaCl, pH 2.5
`
`50 mM glycine /0.1 mM NaCl, pH 2.5
`
`60 +6
`
`8349
`
`2 M guanidine-HC1/0.15M NaCl, pH2.5
`
`98+1
`
`0.025% Trypsin
`
`3541
`
`to
`“T84 cells were incubated with 10 nM ['5I]ST at 4°C for 2 h,
`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.
`® % ["*5]]ST dissociated from cell surface receptors = (radioactivity asso-
`ciated with cells after incubation with dissociating agent)/(radioactivity
`associated with cells before incubation with dissociating agent).
`* Dissociation of ['?°IJST from celis with trypsin was performed as
`described in Section 2.
`
`Time (hour)
`
`Fig. 3. Comparison of the binding of ['°1]ST to T84 cells at 4°C
`(squares) and 37°C(circles). Specific binding of radioligand to T84cells
`at different temperatures was quantified as described in Section 2 and
`Figs.
`1 and 2. Data are representative of at least three experiments. Error
`bars, S.E.
`
`3 h incubation in the presence of ['?°IJST, once equilib-
`rium was achieved. These observations demonstrate that
`internalization of ['7>I]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 of ['*°1]ST to T84 cells at 37°C
`
`Total radioactivity reflecting the association of ['?°IJST
`with T84 cells increased in a time-and concentration-de-
`pendent fashion upon incubation of these cells with in-
`creasing 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. Further-
`more, cell-associated radioactivity could not be detected
`when the highest concentration of ['*°I]ST (107* M) was
`incubated with COS-7 cells, which do not express GCC
`receptors, for 8 h [23].
`Total radioactivity associated with T84 cells resulting
`from their incubation with ['?°IJST at 37°C could reflect
`ligand boundto cell surface receptors and/or radioactivity
`in the intracellular compartmentreflecting internalized lig-
`and—receptor complexes. ST associated with surface recep-
`tors was quantified at various times by incubating T84
`cells with acidic guanidine-HCl, as described above (Fig.
`2B). ST boundto cell surface receptors in a concentration-
`and time-dependent and saturable fashion. Cell surface
`binding appeared to reach equilibrium at 37°C in a concen-
`tration-independent fashion, with equilibrium achieved by
`60 min with all ligand concentrations examined (data not
`shown). ['5I]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 concentra-
`tions studied (10~* M).
`
`

`

`R. Urbanski et al. / Biochimica et Biophysica Acta 1245 (1995) 29-36
`
`33
`
`»
`
`NSwe
`
`
`
`
`
`(Lnfroles/106celts)
`
`50 w
`
`LinofRadiolabeledSTBoundto
`
`theCeSurfaceorFree
`
`
`
`%ofCell-AssociatedRadiolabeledSTRecovered
`Intracellularly
`
`g
`
`10
`
`tration of GCC at the surface of these cells is temperature-
`and ligand-independent. To examinethis further, T84 cells
`were incubated with near-saturating concentrations (10
`nM)of ['?°I]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 ceil surface receptors was time-depen-
`dent 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~* M,calculated by double reciprocal
`plot analysis of the time course of binding of ['?°I]ST to
`cell
`surface receptors, was
`similar at 37°C (5043
`fmol/10° cells) and 4°C (55+7 fmol/10° cells). As
`demonstrated above, there is no detectable internalization
`of ['*°I]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
`
`100
`
`%ofRadiolabeledSTAssociatedwiththeCellorFreeintheMedia
`LnofRadiolabeledSTBoundtoCell
`
`SurfaceReceptors{Lnfmoles/106
`cells)
`
`75
`
`50
`
`25
`
`»
`
`°
`
`20
`
`40
`
`60
`
`Time (min)
`
`Fig. 4. Kinetics of ['*I]ST bound at 4°C to T84 cells which were
`subsequently increased to 37°C. T84 cells (10° cells/well) were incu-
`bated with 10 nM ['*4I]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 radioac-
`tivity 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.
`
`Time (min)
`
`Fig. 5. Kinetics of internalization of ['71]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 boundto 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-regu-
`lation at the cell surface, following receptor endocytosis
`has been observed previously [11,12,17,26—32].
`
`3.6. Kinetics of surface-bound ['*?IJST in T84 cells
`
`The fate of ['?°IJST bound to surface receptors in T84
`cells was directly examined.
`In these experiments, cell
`surface ST receptors were pre-bound with ['IJST by
`incubating T84 cells with 10 nM ['**IJST at 4°C for 2.5 h.
`At this temperature, there is no internalization of radiolig-
`and 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
`['°I]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 decreasein cell-associated ['**IJST within the first 9
`min, to about 30% ofthat 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 and/or receptor populations
`contributing to different rates of loss of ST from cells (Fig.
`4B). The initial rapid rate of loss of cell-associated ra-
`dioactivity 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 min)rate ofloss of cell-associated radioac-
`tivity is very slow and occurs in the absence ofinternaliza-
`
`

`

`34
`
`R. Urbanskiet 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 previ-
`ously 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-re-
`ceptor complexes and their functional significance remain
`unclear.
`
`3.7. Kinetics of ['?°IST internalization in T84 cells
`
`['°I]ST increasedin the intracellular compartmentin 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 de-
`crease 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 bind-
`ing 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 and/or 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).
`Therate of loss of cell surface radioactivity reflects disso-
`ciation of ligand-receptor complexes into the media and
`endocytosis of those complexes. Therefore,
`int
`dissoc
`Kegse =k +k
`
`where k,,.. is the rate constantof loss of radioactivity from
`dissociablesites at the cell surface, k,,,
`is the rate constant
`of internalization, and ky,;,,,. is the rate constant of dissoci-
`ation 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 semilogarith-
`mic plot of the time course of loss of cell surface radioac-
`tivity (Fig. 4B). The rate constant of dissociation of lig-
`and—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 radioac-
`tivity from the cell surface and of dissociation, estimated
`from the
`slopes of
`the
`semilogarithmic plots, were
`koe = 0.42 £0.08 min@!s kgissoo = 0-14 + 0.01 min™', re-
`spectively. Employing these values, the calculated rate of
`internalization of GCC was: k,,, = 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 re-
`ceptors [33].
`
`4. Discussion
`
`The present studies demonstrate that ST in human
`colonic tumorcells in vitro undergo ligand-induced recep-
`tor-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,
`surface GCC molecules do not
`suggesting that cell
`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 eluci-
`dated [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 suggestthat
`it is only the dissociable sites that undergo endocytosis.
`Endocytosis of GCC appears to be a rapid event, compara-
`ble 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 charac-
`teristics 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 ob-
`served to be about 70% of that noted at 37°C. These
`differences are consistent with the present observations
`that at 4°C the amountof radioactivity associated with T84
`cells reflects ligand boundto 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 temper-
`atures 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 endo-
`cytosis of ligand-receptor complexes [15].
`
`

`

`R. Urbanskiet al. / Biochimica et Biophysica Acta 1245 (1995) 29-36
`
`35
`
`receptor-coupled guanylyl
`Endocytosis of peptide
`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 re-
`ceptors [13]. Rather, signal
`termination in this system
`appeared to be mediated by a very rapid dissociation of
`ANPfrom receptors. This rapid dissociation of ligand from
`GCAis 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~! [21]. These observations
`suggest
`that rapid dissociation of ligand-receptor com-
`plexes may play a prominentrole in signal termination by
`guanylyl cyclase-coupled receptors [13]. The precise mech-
`anisms underlying acceleration of the dissociation rate of
`these receptors in intact cells compared to cell-free mem-
`branes remain to be elucidated.
`In contrast
`to GCA in renal cells, GCB undergoes
`ligand-dependentinternalization in PC12 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
`GCCin 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 subse-
`quently measuring cell surface receptors remaining by
`incubation with labeled ligand.
`In these studies,
`if the
`dissociation rate of ligand—receptor complexes is not suffi-
`ciently 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-regu-
`lation 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 recy-
`cling 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].
`is
`Ligand-induced receptor-mediated internalization
`first-order with respect to the concentration of the ligand—
`receptor complexat the cell surface [18,35]. Typically, the
`rate of ligand-induced receptor internalization can be esti-
`mated directly in experiments in which cell surface recep-
`tors are preloaded in the absence ofinternalization 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
`
`in order for the
`lower than the rate of internalization,
`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 internaliza-
`tion. 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). Radioac-
`tivity 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 internaliza-
`tion. Although the estimate for the rate of internalization of
`GCC presented herein is only a lower limit,
`it demon-
`strates that these peptide receptors undergo rapid internal-
`ization 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
`CFTR [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 internal-
`ization of GCC. Endocytosis is initiated by association of
`ST with the extracellular ligand binding domain of GCC.
`Ligand-receptor interaction may inducealterations 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 mes-
`senger, cGMP,uponligand binding [11,12,17,24]. Further-
`more,
`ligand-induced receptor-mediated endocytosis de-
`pends on specific interactions between cytoplasmic do-
`mains of receptors and components of the endocytic appa-
`ratus [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 endocyto-
`sis
`[38,39].
`Indeed,
`receptors which undergo ligand-
`induced receptor mediated endocytosis, such as those for
`epidermal growth factor, insulin-like growth factor I], and
`vesicular stomatitis virus glycoprotein G, possess in their
`
`

`

`36
`
`R. Urbanski et al. / Biochimica et Biophysica Acta 1245 (1995) 29-36
`
`cytoplasmic domains the consensus sequence YXXZ [39-
`42]. Of significance,
`this sequence also appears in the
`carboxy-terminal region of GCC, supporting the sugges-
`tion that endocytosis of this receptor is mediated by mech-
`anisms involving coated pits.
`Once GCCis incorporated into coated pits, Jigand—re-
`ceptor 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 endoso-
`mal 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 Manufac-
`turers 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, LK., Buxton