Hormones and their Actions
`Part I
`
`Editors
`
`B.A. COOKE
`Department of Biochemistry, Royal Free Hospital School of Medicine, University
`of London, Rowland Hill Street, London NW3 2PF, England
`
`R.J.B. KING
`Hormone Biochemistry Department, Imperial Cancer Research Fund
`Laboratories, P.O. Box No. 123, Lincoln's Inn Fields,
`London WC2A 3PX, England
`
`H.J. van der MOLEN
`Nederlandse Organisatie voor Zuiver-Wetenschappelijk Onderzoek.(Z.W.O.
`Postbus 93138, 2509 AC Den Haag, The Netherla.ids
`
`igc9
`
`$
`
`1988
`ELSEVIER
`Amsterdam • New York • Oxford
`
`MYLAN EXHIBIT - 1013
`Mylan Pharmaceuticals, Inc. v. Bausch Health Ireland, Ltd. - IPR2022-00722
`
`

`

`© 1988, Elsevier Science Publishers B.V. (Biomedical Division)
`
`All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or trans-
`mitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without
`the prior written permission of the Publisher, Elsevier Science Publishers B.V. (Biomedical Division),
`P.O. Box 1527, 1000 BM Amsterdam, The Netherlands.
`
`No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a
`matter of products liability, negligence or otherwise, or from any use or operation of any methods, prod-
`ucts, instructions or ideas contained in the material herein. Because of the rapid advances in the medical
`sciences, the Publisher recommends that independent verification of diagnoses and drug dosages should
`be made.
`
`Special regulations for readers in the USA. This publication has been registered with the Copyright
`Clearance Center, Inc. (CCC), Salem, Massachusetts. Information can be obtained from the CCC about
`conditions under which the photocopying of parts of this publication may be made in the USA. All other
`copyright questions, including photocopying outside of the USA, should be referred to the Publisher.
`
`ISBN 0-444-80996-1 (volume)
`ISBN 0-444-80303-3 (series)
`
`Published by:
`
`Sole distributors for the USA and Canada:
`
`Elsevier Science Publishers B.V.
`(Biomedical Division)
`P.O. Box 211
`1000 AE Amsterdam
`The Netherlands
`
`Elsevier Science Publishing Company, Inc.
`52 Vanderbilt Avenue
`New York, NY 10017
`USA
`
`Library of Congress Cataloging in Publication Data
`Hormones and their actions / editors, B.A. Cooke, R.J.B. King, H.J. van der Molen.
`p. cm. -- (New comprehensive biochemistry; v. 18A-)
`Includes bibliographies and index.
`ISBN 0-444-80996-1 (pt. 1)
`1. Hormones--Physiological effect. I. Cooke, Brian A. II. King, R.J.B. (Roger John Benjamin) III.
`Molen, H.J. van der.
`IV. Series: New comprehensive biochemistry; v. 18A, etc.
`[DNLM: 1. Hormones--physiology. WI NE372 v. 18 / WK 102 H812781
`QD415.N48 vol. 18A, etc.
`[QP5711
`574.19'2 s--dc 19
`[612'.4051
`DNLM/DLC
`for Library of Congress
`
`88-16501
`CIP
`
`Printed in The Netherlands
`
`

`

`B.A. Cooke. R.J.B. King and H.J. van der Molcn (eds.)
`Hormones and their Actions, Part I
`O 1988 Elsevier Science Publishers BV (Biomedical Division)
`
`133
`
`CHAPTER 9
`
`Internalization of peptide hormones and
`hormone receptors
`
`DEBORAH L. SEGALOFF and MARIO ASCOLI
`
`The Population Council, 1230 York Avenue, New York, NY 10021, U.S.A.
`
`1. Introduction
`
`Peptide hormones are one class of many agents present in the bloodstream that af-
`fect the multiplication and differentiated functions of mammalian cells. The ability
`of a particular peptide hormone to elicit an effect in the appropriate target cell is
`dictated by the presence of receptors on the surface of the target cell which specif-
`ically bind that hormone. Although the cellular responses to the different peptide
`hormones vary, as do many of the mechanisms of signal transduction that translate
`the binding of the hormone to the cellular response, there is one salient feature that
`all peptide hormones studied to date share. This is the receptor-mediated endocy-
`tosis (RME) of the hormone.
`The idea that proteins could be internalized by a receptor-mediated mechanism
`by their target cells was sparked by the pioneering studies of Goldstein and co-
`workers [1] and by Cohen and co-workers [2,3], who obtained evidence for the re-
`ceptor-mediated internalization and degradation of low-density lipoprotein (LDL)
`and epidermal growth factor (EGF), respectively, in the mid 1970s. Although en-
`docytosis of a non-specific nature had been described by then, the concept of en-
`docytosis of a specific ligand being mediated by the binding of that ligand to a cell
`surface receptor was unprecedented.
`These investigators were one of the first to study the binding of '25I-labelled li-
`gands to intact cells (as opposed to studying the binding of the ligand to mem-
`branes, which was the prevailing approach at the time). Interestingly, their studies
`showed that when the binding studies on the cultured cells were performed at 37°C,
`but not at 4°C, there was a time-dependent accumulation of degradation products
`
`Abbreviations and trivial names used are: RME, receptor-mediated endocytosis; LDL, low density li-
`poprotein; EGF, epidermal growth factor; SDS, sodium dodecyl sulfate; LH, luteinizing hormone; hCG,
`human chorionic gonadotropin; and G protein, guanine nucleotide binding protein.
`
`

`

`134
`
`of the ligand in the culture medium. That the degradation of these ligands was oc-
`curring as a result of internalization of the ligand into the cell was suggested by ob-
`servations that the accumulation of degradation products in the medium was both
`energy- and temperature-dependent and that it could be inhibited by agents known
`to inhibit lysosomal function. By using specific treatments to release the surface-
`bound [125I]LDL or [125I]EGF, it was possible to document the appearance of in-
`tracellular radioactivity (representing intact or partially degraded ligand) prior to
`the release of degradation products into the medium. Furthermore, it was found
`that some compounds (such as metabolic inhibitors) prevented the accumulation of
`intracellular ligand (presumably by inhibiting internalization); whereas other com-
`pounds known to inhibit lysosomal function (such as NH4C1 or chloroquine) al-
`lowed internalization, but prevented degradation of the ligand [3-7].
`Concomitant morphological studies by electron microscopy on the fates of re-
`ceptor-bound LDL and EGF (using ligands covalently attached to electron-dense
`ferritin) elegantly confirmed the inferences from the biochemical data that these li-
`gands were internalized and degraded in the lysosomes [8-11]. Since the internal-
`ization and degradation of ligand was strictly dependent upon binding of the ligand
`to the cell surface receptor, this process was called receptor-mediated endocytosis
`(RME).
`RME has since been shown to occur with other transport proteins, other growth
`factors, and with peptide hormones (for reviews see Refs. 12-16). The general fea-
`tures of RME as they are understood today from biochemical and morphological
`studies on a variety of ligands are discussed below as they pertain to peptide hor-
`mones.
`
`2. General features of receptor-mediated endocytosis
`
`A schematic overview of RME is shown in Fig. 1. The cell surface receptors for a
`particular hormone are either located in areas of the plasma membrane referred to
`as coated pits or they are randomly distributed throughout the cell surface and mi-
`grate to the coated pits upon binding of the hormone. Coated pits are indented areas
`of the plasma membrane where there is an intracellular `lining' of the membrane
`with the protein clathrin and they constitute a small percentage (<5%) of the total
`area of the plasma membrane [8,17,18]. In the cases where the hormone-receptor
`complexes migrate to coated pits, there often is a microaggregation of the com-
`plexes (two to four per group) during this redistribution [19]. Following this micro-
`aggregation there is a more masssive clustering of hormone-receptor complexes in
`the coated pits.
`Coated pits containing receptor-bound hormones become invaginated and pinch
`off intracellularly to form what are called coated vesicles. The coated vesicles still
`have clathrin associated with them, forming basket-like structures around the ves-
`
`

`

`135
`
`coated pit
`
`coated
`vesicle
`
`receptor
`sequestered
`
`receptor
`recycled
`
`or
`
`\
`endosome
`
`•
`0 •
`•
`CURL
`
`om,
`
`0
`
`lysosome
`
`-7
`
`hormone and receptor
`degraded
`
`'%
`
`•
`
`•
`
`•
`
`•
`
`•
`hormone
`degraded
`
`y receptor
`hormone
`X clathrin
`
`Fig. 1. Schematic representation of the possible routes of receptor and hormone during RME.
`
`icles [20]. The lumen (fluid-filled interior) of the coated vesicles does not have any
`free hormone. At this stage, the hormone is still bound to the receptor, facing the
`lumen [10]. With time, the coated vesicles shed their clathrin coats and fuse with
`other similar vesicles; all this time these vesicles are moving further into the interior
`of the cell [15]. The prelysosomal vesicles resulting from these fusions are called
`endosomes or endocytic vesicles and have a critical role in RME due to the acidic
`environment of their lumen.
`Although not as acidic as lysosomes (with an intra-compartmental pH of 4.5, see
`Ref. 21), the pH 5.5 environment of the endosome [22] is sufficiently low to cause
`the dissociation of some hormones from their receptors. When this occurs, there is
`a subsequent sequestering of the free hormone from the receptor in a related ves-
`icle and tubule compartment called CURL (compartment for uncoupling of recep-
`tor from ligand, see Ref. 23), where the free hormone is sequestered into the ves-
`icular structure while the receptor accumulates in the membrane of the tubule
`structure. A subsequent physical separation of these compartments allows for the
`differential processing of the hormone versus the receptor. Thus, while the free
`hormone is ultimately delivered (via vesicle fusion) to the lysosome where it is de-
`graded, the free receptor may be recycled (via the Golgi compartment) to the cell
`
`

`

`136
`
`surface, where it can rebind hormone and repeat the whole process of RME. The
`free receptor may also remain sequestered intracellularly.
`It should be pointed out, however, that not all hormones dissociate from their
`receptor in the pH 5.5 environment of the endosome [24]. Some hormone-receptor
`complexes require much lower pH values for dissociation to occur. Although not a
`peptide hormone, the iron-transport protein transferrin is a peculiar example of this
`phenomenon and should be pointed out. In this case, at the neutral pH of the ex-
`tracellular fluid transferrin containing bound iron binds to its cell surface receptor
`and is internalized. In the low pH environment of the endosome, iron becomes dis-
`sociated from transferrin, but transferrin remains bound to its receptor. The trans-
`ferrin receptor, with bound transferrin, is then recycled to the cell surface. With
`iron no longer bound to the transferrin, the transferrin readily dissociates from its
`receptor at the neutral pH of the extracellular fluid [25,26]. This mechanism pro-
`vides for an efficient continual uptake of iron into cells. Unlike transferrin, how-
`ever, in those instances where peptide hormones have been documented not to be
`dissociated from their receptor in the endosome compartment, the hormone and
`receptor are delivered to the lysosomes via fusion of the endosomes with lyso-
`somes, where both hormone and receptor are degraded [24,27]. The continuous
`degradation of the receptor with each round of RME eventually leads to a decrease
`in the number of receptors on the cell surface, a phenomenon called down-regu-
`lation.
`The distinction between a given receptor being recycled versus degraded is not
`always an all-or-none phenomenon. In fact, in many cases both processes occur to
`different degrees. Thus, even though the majority of receptors may be recycled,
`each round of endocytosis can result in the degradation of a small percentage of
`receptors. If the rate of synthesis of new receptors plus the rate of recycling of in-
`ternalized receptors is slower than the rate at which receptors are degraded with
`each round of RME, there will eventually be a down-regulation of the cell surface
`receptors. Another factor to be taken into account is that some receptors may be
`spared degradation, but they may not be immediately recycled back to the cell sur-
`face (i.e., they may be sequestered intracellularly). These possible routes of recep-
`tor disappearance and appearance on the cell surface are summarized schematically
`in Fig. 2.
`Whether a given hormone receptor is recycled or not during RME depends not
`only upon which hormone the receptor binds, but also upon the cell type and stage
`of differentiation of a given cell. Thus, the insulin receptor has been shown to be
`recycled during RME in rat adipocytes [28,29], but not in lymphocytes [30]; and it
`is down-regulated in the adult rat liver [31], but not in the fetal rat liver [31].
`
`

`

`137
`
`recycling
`
`/
`
`internalization—. sequestration
`(intracellular pool)
`
`degradation
`
`t
`synthesis
`
`Fig. 2. Possible routes of receptor appearance and disappearance from the cell surface.
`
`3. Methods used to assess receptor-mediated endocytosts
`
`3.1. Morphological approaches
`
`On a light microscopic level it is possible to visualize the binding of fluorescently
`labelled hormones to intact cells or to visualize the native hormone with fluorescent
`antibodies [32-35]. Using fluorescently labelled hormones, investigators have ob-
`served a band of fluorescence defining the circumference of each cell when the
`binding of the fluorescently labelled hormone to the cells was performed under
`conditions where internalization was inhibited (such as at 4°C). When the cells were
`allowed to bind hormone at 4°C, washed to remove unbound hormone, and then
`incubated at 37°C to allow the surface-bound hormone to be internalized, it was
`possible to observe a concentration of the fluorescence into small patches on the
`cell surface and a subsequent increase in diffuse fluorescence located inside the cell.
`This experimental approach is powerful in that it allows one to visually determine
`whether under different conditions a hormone is bound to the cell surface or is in-
`ternalized, and therefore it has been widely used. In order to identify the particular
`organelles with which the internalized hormone becomes associated, however, it is
`necessary to examine the cells using an electron microscope.
`Using electron microscopy, one can `follow' the fate of a given peptide hormone
`in its target cell by using preparations of hormone that have been coupled to elec-
`tron dense particles, such as ferritin or colloidal gold; or by using hormone prep-
`arations that have been radiolabelled to a high specific activity (typically with 125I)
`and performing autoradiography [8,9,11]. Alternatively, one can bind the unal-
`tered hormone to the cell, prepare the sample for electron microscopy and then bind
`an electron-dense anti-hormone antibody to the sample to visualize the hormone
`[23]. The latter approach is generally preferable in that one need not be concerned
`that the electron dense or radiolabelled hormone is handled by the cell differently
`than the native hormone. Since colloidal gold is available in a range of sizes, if an
`antibody to the receptor is available (that can recognize the receptor even when
`hormone is bound to it), then by using an anti-receptor antibody coupled to col-
`loidal gold of one diameter and an anti-hormone antibody couple to colloidal gold
`of a different diameter, one can simultaneously follow the fate of both the hormone
`and the receptor during RME [23].
`
`

`

`138
`
`Using these approaches, it has been found that when the binding of the hormone
`to the cells is done at 4°C the hormone is associated with the plasma membrane
`only. If the binding is done at 4°C, the cells washed to remove unbound hormone
`and then subsequently warmed to 37°C, there is a decrease in the cell surface-bound
`hormone and a concomitant increase in intracellular hormone. By morphological
`appearances and by enzymatic or immunological staining, it is possible to identify
`the intracellular compartments with which the hormone is associated. Further-
`more, if one uses a hormone (or antibody to the hormone) made electron dense,
`the resolution is usually fine enough that one can assess whether the hormone is
`associated with the organelle membrane (and thus probably receptor-bound) or is
`free in the lumen [10].
`Typically, a morphometric analysis is performed where a large number of micro-
`graphs taken at each time point are examined and the number of grains of ferritin
`or gold particles associated with a given cellular organelle (plasma membrane, coated
`vesicle, endosome, lysosome, etc.) are tabulated. As such, one can calculate the
`percentage of grains or particles associated with a given organelle at each time point
`and arrive at a statistically valid conclusion as to the route of the hormone (and/or
`receptor) during RME [9,11,36].
`
`3.2. Biochemical approaches
`
`In order to study the RME of a peptide hormone biochemically, it is necessary to
`be able to radiolabel the hormone to a high specific activity with 125I, while retain-
`ing the normal binding and biological properties of the hormone. As discussed in
`the introduction, if one binds the iodinated hormone to intact cells at 37°C and de-
`tects ligand degradation products in the medium, that is an indication that RME of
`the hormone may be occurring. Ligand degradation can be ascertained by analyz-
`ing the molecular size of the radioactive products by gel filtration or by testing the
`precipitability of the radioactivity by trichloroacetic acid [37]. Since single amino
`acids and small peptides are not precipitable by trichloroacetic acid, the percentage
`of acid-soluble radioactivity in the medium represents the percentage of degraded
`ligand. It is then necessary to document that the accumulation of acid-soluble ra-
`dioactivity in the medium is dependent upon the extent of hormone binding, the
`length of the incubation, and the temperature (such that degradation of the hor-
`mone should not be apparent at 4°C). Furthermore, one should be able to inhibit
`the appearance of acid-soluble radioactivity with metabolic inhibitors (such as NaN3)
`or with compounds that inhibit the delivery of the hormone to the lysosomes or in-
`hibit lysosomal function (such as leupeptin,
`chloroquine, or monensin; see
`Refs. 38,39).
`When one measures the amount of hormone bound to an intact cell at 37°C, this
`represents a sum of surface-bound hormone plus hormone that has since been in-
`ternalized (and is in an intact or partially degraded form). It should be noted that
`
`

`

`139
`
`once an internalized protein has been degraded to free amino acids, these are rap-
`idly released from the cell, and thus are not detected to an appreciable extent within
`the cell. In order to measure the level of surface-bound versus internalized hor-
`mone, it is necessary to develop a method that will quantitatively release the sur-
`face-bound hormone. Many peptide hormones can be dissociated from their recep-
`tor under conditions of low pH (pH 3-4) and thus this has been a commonly used
`method [6,36]. An advantage of this method is that it is a mild treatment and thus
`in some cases one can treat the cells with acid to remove the surface-bound hor-
`mone and then rebind fresh hormone and observe a cellular response [36]. Another
`method that is generally applicable is to degrade the surface-bound hormone by
`adding proteases using conditions that do not lyse the cells or allow penetration of
`the added enzyme [3]. It should be noted, however, that this treatment may also
`damage the receptor and thus cannot be used if one wishes to subsequently rebind
`fresh hormone to the cells. Lastly, a variety of other methods tailored to the bind-
`ing characteristics of a given ligand have also been used [5,40,41]. With any given
`treatment, however, it is necessary to document that one is indeed releasing most
`(or all) of the surface-bound hormone. This can be done by saturating the binding
`sites of the cell with radiolabelled hormone under conditions where no internali-
`zation should occur (such as at 4°C) and then testing if the treatment releases all
`the cell-associated radioactivity. Thus, by measuring total cell-associated radioac-
`tivity in one set of cells and releasable radioactivity in another set of cells, one can
`calculate the amount of internalized hormone by subtraction. Therefore, one can
`in fact measure hormone binding to an intact cell at 37°C and construct a time course
`of cell surface-bound hormone, internalized hormone and degraded hormone. Un-
`der these conditions, cells are continuously exposed to hormone in the medium and
`thus are undergoing many rounds of RME. If the internalized receptor is not re-
`cycled back to the cell surface, the cell surface receptor will become down-regu-
`lated. A schematic example of a time course of hormone binding and internaliza-
`tion to intact cells where the receptor is down-regulated is shown in Fig. 3A. In
`contrast, Fig. 3B depicts a representation of such a time course when the internal-
`ized receptor is not down-regulated. It should be pointed out that the maximal
`amount of hormone internalized and/or degraded will vary depending upon the ex-
`tent of receptor recycling. Thus, if the receptors do not recycle, the maximal amount
`of hormone internalized and/or degraded should be less than or equal to the num-
`ber of cell surface receptors. Therefore, the amount of hormone that is processed
`in this case is dictated by the number of hormone receptors. If the receptors do re-
`cycle, then the maximal amount of hormone internalized and/or degraded should
`exceed the number of cell surface receptors. In this case the cells can theoretically
`degrade all the added hormone, regardless of the number of hormone receptors.
`From the biochemical approaches discussed thus far, one can conclude that a given
`hormone may be internalized by RME. Conclusive evidence for such internaliza-
`tion, however, can only be obtained by concurrent morphological data as described
`
`

`

`surface -bound
`
`Internalized
`
`B
`
`/
`/
`
`—I-
`
`infernalrzed
`
`--
`
`--
`
`1'
`
`— degraded
`
`degraded
`
`Surface - bound
`
`140
`
`'251-HORMONE
`
`LENGTH OF OF INCUBATION (hours)
`
`Fig. 3. Distribution of hormone bound to cells during many rounds of endocytosis. Cells are incubated
`with radiolabelled hormone for increasing lengths of time at 37°C. At various time points, hormone that
`is surface-bound, internalized or degraded and released into the medium is determined as described in
`the text. Panel A represents a case where the cell surface receptor becomes down-regulated; Panel B
`represents a case where the cell surface receptor is not down-regulated, but instead is recycled.
`
`above. For data on the rate of internalization of the hormone and possible down-
`regulation and/or recycling of the receptor one must again use biochemical ap-
`proaches.
`A commonly used method to calculate the rate of internalization of a hormone
`is to bind the hormone to intact cells at 4°C (where no internalization should oc-
`cur), wash the cells to remove unbound hormone and then measure the amount of
`surface-bound radioactivity remaining as a function of time after warming the cells.
`Unlike the experimental approach described above where the cells are allowed to
`continuously bind and internalize hormone at 37°C and thus undergo many rounds
`of RME (see Fig. 3), under these conditions the cells are internalizing only the pre-
`bound hormone and thus are undergoing only one round of RME. A schematic
`example of results of this kind of experiment is shown in Fig. 4. Typically, one ob-
`serves a loss of surface-bound radioactivity with a concomitant increase in the levels
`of internalized radioactivity. Since the internalized hormone is degraded, the levels
`of internalized radioactivity subsequently decline and there is an increase in the
`levels of degradation products in the medium. It should be noted that when these
`experiments are done it is difficult to detect a lag in the appearance of the inter-
`nalized radioactivity; however, there is a lag in the appearance of degradation
`products in the medium [36]. This lag is a composite of the rate of accumulation of
`hormone in the lysosomes, the rate of hormone degradation and the rate of release
`of degradation products. Among these processes, the rate of hormone degradation
`appears to be limiting [36]. The use of the loss of cell surface-bound radioactivity
`as a measure of the rate of hormone internalization is valid, though, only when there
`is little or no dissociation of the hormone from the receptor during the 37°C incu-
`bation (which can be assessed by the appearance of trichloroacetic acid-insoluble
`radioactivity in the medium). Otherwise, the rate of loss of surface-bound hormone
`would reflect both the rate of internalization of receptor-bound hormone and the
`rate of dissociation of the hormone from the cell surface receptor [42].
`
`

`

`141
`
`surface-bound
`
`internalized
`
`PC
`/ ,
`/ degraded
`
`TIME AFTER WARMING (minutes)
`
`Fig. 4. Distribution of hormone bound to cells during one round of endocytosis. Cells are incubated with
`hormone at 4°C to saturate the cell surface receptor. At 1=0, the cells are washed to remove unbound
`hormone and warmed to 37°C. At various time points after warming, the hormone that is surface-bound,
`internalized, or degraded and released into the medium is determined as described in the text.
`
`A more valid approach to calculating the rate of internalization of a hormone is
`to use a steady-state approach as originally described by Wiley and Cunningham
`[43-45]. In this approach, the cells are incubated with the hormone at 37°C under
`conditions where the cells undergo many rounds of RME as they continuously bind
`and internalize hormone (c.f., Fig. 3). Under these experimental conditions, the
`rate of internalization can be calculated from the ratio of internalized to surface-
`hound hormone provided that (i) the time course chosen is shorter than the ob-
`served lag of appearance of degradation products in the medium (see above); and
`(ii) the level of surface-bound radioactivity is at a steady state [43]. Although the
`first of these two criteria must always be met, one can also perform this experiment
`while the surface-bound radioactivity is approaching a steady state. If this is done,
`however, the rate of internalization is calculated from the ratio of the internalized
`radioactivity (which is, by definition, an integral since the experiment is done be-
`fore any degradation products are released into the medium) versus the integral of
`the surface bound radioactivity [42,44,45]. In addition to the rate of internalization,
`the steady-state analysis described by Wiley and Cunningham allows one to calcu-
`late many other parameters pertaining to the hormone-receptor interaction during
`RME. These include the steady-state association constant for the hormone-recep-
`tor complex (a steady state equivalent of the Ka calculated by Scatchard analysis),
`the number of cell surface receptors, the rate of receptor appearance at the cell sur-
`face, the rate constant for the internalization of occupied receptors, the rate con-
`stant for the internalization of unoccupied receptors and the rate constant for the
`degradation of the internalized hormone. Furthermore, if the receptor for the hor-
`
`

`

`142
`
`mone is down-regulated, one can use this steady-state model to determine if the
`down-regulation is due to an increase in the rate of internalization of occupied ver-
`sus unoccupied receptors or to a decrease in the rate of appearance of receptors on
`the cell surface. Using this approach, both hCG and EGF have been shown to down-
`regulate their respective receptors by increasing the rate of internalization of the
`occupied versus the unoccupied receptor [44,46].
`Another important aspect of RME that one would want to determine is the in-
`tracellular route of the hormone and receptor. As discussed above, one can use
`morphological approaches to address this question. The morphological approach is
`particularly elegant if one has an antibody to the receptor such that one can simul-
`taneously detect both the hormone and its receptor. One can, however, also ad-
`dress this question biochemically. Indeed, it is possible to fractionate cell extracts
`on Percoll gradients into fractions composed primarily of plasma membrane, en-
`dosomes or lysosomes [24,47,48]. Thus, one can bind radiolabelled hormone to the
`cells, allow the cells to internalize the hormone for a given length of time, and then
`fractionate the cells to determine in which intracellular compartment the hormone
`(i.e., radioactivity) is located. One can determine whether the internalized hor-
`mone is free or receptor-bound by precipitation of the internalized radioactivity by
`polyethylene glycol or ammonium sulfate [24,49]. Furthermore, by analyzing the
`internalized radioactivity in the different compartments on SDS-polyacrylamide gels,
`one can assess whether the hormone is intact or partially degraded [24,48]. Thus,
`one can determine in which compartment the hormone dissociates from its receptor
`and in which compartment degradation of the hormone occurs. Using these tools,
`it has been possible to document that unlike many other hormones which dissociate
`from their receptor in the endosome, hCG remains receptor bound. Thus, the hCG-
`receptor complex is delivered to the lysosome intact, whereupon the complex is dis-
`sociated [24]. Although it can only be directly ascertained that the hormone is then
`degraded (since it is the hormone which is radiolabelled), it is assumed that delivery
`of the hCG receptor to the lysosome also results in its degradation (which is con-
`sistent with the down-regulation of the hCG receptor in these cells).
`Another frequently used tool to assess the intracellular route of internalized hor-
`mones and their receptors is the use of compounds or conditions that allow hor-
`mone binding and internalization to occur, but impede the intracellular route of the
`internalized hormone receptor. By using an inhibitor of lysosomal enzymes, such
`as leupeptin, one can `trap' undegraded hormone (and possibly receptor) in the ly-
`sosome [24]. By performing the experiment at 16-20°C, it is possible to internalize
`receptor-bound hormone, but `trap' it in the endosome compartment [50]. Other
`compounds such as monensin and NH4C1 can be used to raise the pH in intracell-
`ular organelles [39]. Unfortunately, since pH gradients exist in both endosomes and
`lysosomes (and other intracellular organelles), these compounds may impede any
`one (or many) of the steps in the transit of the hormone and receptor, and thus one
`must use additional approaches (as outlined above) to determine in which organelle
`
`

`

`143
`
`the hormone (or receptor) has been trapped. Thus, although it has been shown that
`in many cases monensin and NH4C1 trap the ligand-receptor complex in the en-
`dosomes [47,48,51], it has been documented that in murine Leydig tumor cells these
`compounds allow the delivery of hCG (bound to its receptor) from the endosome
`to the lysosome but inhibit the subsequent dissociation of hCG from its receptor
`and degradation of the hormone [24].
`Once the hormone-receptor complex has been internalized, the receptor may be
`degraded, sequestered intracellularly, and/or recycled back to the cell surface. If
`the receptor were sequestered intracellularly, then one should be-'4;le to allow cells
`to internalize hormone and then detect a pool of intracellular receptors in a deter-
`gent extract of the cells. To do this, one would allow the cells to bind and inter-
`nalize unlabelled hormone and then measure the binding of radiolabelled hormone
`to the intact cells versus a detergent extract of the cells (where both the cells and
`extract have been treated with acid to remove the unlabelled hormone prior to add-
`ing the radiolabelled hormone). Since the detergent extract would be composed of
`both cell surface and intracellular receptors, an increase in binding activity of the
`detergent extract and a decrease in binding to the intact cells would be indicative
`that the receptors internalized during RME of the unlabelled hormone were being
`sequestered intracellularly. Alternatively, if one detected a decrease in the binding
`activity in the intact cell and in the detergent extract, this would indicate that