ef OL)" ree
`
`TRAD
`
`BOCK¥enaANGtiewi bo
`
`Leck
`
`Hormonesand 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.O27
`Postbus 93138, 2509 AC Den Haag, The Netherlands
`
`
`
`1988
`ELSEVIER
`Amsterdam - New York - Oxford
`
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`

`

`© 1988, Elsevier Science Publishers B.V. (Biomedical Division)
`
`All rights reserved. Nopart 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.
`
`Noresponsibility is assumed by the Publisher for any injury and/or damage to personsor property as a
`matter of productsliability, negligence or otherwise, or from anyuse or operation of any methods, prod-
`ucts, instructions or ideas contained in the material herein. Becauseof the rapid advancesin the medical
`sciences, the Publisher recommends that independentverification 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 photocopyingofparts of this publication may be madein 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:
`
`Elsevier Science Publishers B.V.
`(Biomedical Division)
`P.O. Box 211
`1000 AE Amsterdam
`The Netherlands
`
`Sole distributors for the USA and Canada:
`
`Elsevier Science Publishing Company, Inc.
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`
`Library of Congress Cataloging in Publication Data
`Hormonesandtheir 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)IIT.
`Molen, H.J. van der.
`IV. Series: New comprehensive biochemistry; v. 18A, etc.
`[DNLM:1. Hormones--physiology. W1 NE372 v. 18 / WK 102 H81278]
`QD415.N48 vol. 18A, etc.
`[QP571]
`574.19'2 s--de 19
`
`[612’.405]
`DNLM/DLC
`for Library of Congress
`
`Printed in The Netherlands
`
`88-16501CIP
`
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`

`B.A. Cooke, R.J.B. King and H.J. van der Molen (eds.)
`Hormonesandtheir Actions, Part 1
`© 1988 Elsevier Science Publishers BV (Biomedical Division)
`
`133
`
`CHAPTER9
`
`Internalization of peptide hormones and
`hormonereceptors
`
`DEBORAHL. SEGALOFF and MARIO ASCOLI
`
`The Population Council, 1230 York Avenue, New York, NY 10021, U.S.A.
`
`1. Introduction
`
`Peptide hormonesare oneclass of many agents present in the bloodstream thataf-
`fect the multiplication and differentiated functions of mammalian cells. The ability
`of a particular peptide hormoneto 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
`hormonesvary, as do manyof the mechanismsofsignal transduction that translate
`the binding of the hormoneto the cellular response, there is one salient feature that
`all peptide hormonesstudied to date share. This is the receptor-mediated endocy-
`tosis (RME) of the hormone.
`Theidea 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 evidencefor 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 oneofthefirst to study the binding of '**I-labelledli-
`gandsto intact cells (as opposed to studying the binding of the ligand to mem-
`branes, which wasthe prevailing approachat 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 densityli-
`poprotein; EGF, epidermal growth factor; SDS, sodium dodecyl sulfate; LH,luteinizing hormone; hCG,
`human chorionic gonadotropin; and G protein, guanine nucleotide binding protein.
`
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`of the ligand in the culture medium. That the degradation of these ligands wasoc-
`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-dependentandthat it could be inhibited by agents known
`to inhibit lysosomal function. By using specific treatments to release the surface-
`bound [!*5I]LDL or [!*IJEGF,it was possible to document the appearance ofin-
`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 knownto inhibit lysosomal function (such as NH,CI 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(usingligands covalently attached to electron-dense
`ferritin) elegantly confirmed the inferences from the biochemical data that theseli-
`gands were internalized and degraded in the lysosomes [8-11]. Since the internal-
`ization and degradationof ligand wasstrictly dependent upon bindingofthe ligand
`to the cell surface receptor, this process was called receptor-mediated endocytosis
`(RME).
`RMEhassince been shownto occur with other transport proteins, other growth
`factors, and with peptide hormones(for reviews see Refs. 12-16). The generalfea-
`tures of RMEasthey are understood today from biochemical and morphouogical
`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 shownin Fig. 1. The cell surface receptors for a
`particular hormoneare either located in areas of the plasma membranereferred to
`as coated pits or they are randomly distributed throughoutthe cell surface and mi-
`grate to the coated pits upon binding of the hormone. Coated pits are indented areas
`of the plasma membrane wherethereis an intracellular ‘lining’ of the membrane
`with the protein clathrin and they constitute a small percentage (<5%) of thetotal
`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 complexesin
`the coatedpits.
`Coatedpits containing receptor-bound hormones becomeinvaginated and pinch
`off intracellularly to form whatare called coated vesicles. The coated vesiclesstill
`have clathrin associated with them, forming basket-like structures around the ves-
`
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`
`
`coated pit
`
`
`ee
`recycled
`
`coated
`vesicle
`
`ope
`receptor
`sequestered
`
`or
`
`135
`
`”
`
`ae
`
`Tee
`
`far
`
`GAD
`
`CURL)
`
`lysosome
`
`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. Atthis stage, the hormoneisstill bound to the receptor, facing the
`lumen[10]. With time, the coated vesicles shed their clathrin coats and fuse with
`othersimilarvesicles; all this time these vesicles are moving further into the interior
`of the cell [15]. The prelysosomal vesicles resulting from these fusions are called
`endosomesor endocytic vesicles and havea critical role in RME dueto the acidic
`environment of their lumen.
`Although notas 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 hormonesfrom their receptors. Whenthis occurs, there is
`a subsequent sequestering of the free hormone from the receptorin a related ves-
`icle and tubule compartmentcalled CURL (compartment for uncoupling of recep-
`tor from ligand, see Ref. 23), where the free hormoneis 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
`hormoneis ultimately delivered (via vesicle fusion) to the lysosome whereitis de-
`graded, the free receptor may be recycled (via the Golgi compartment)to the cell
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`surface, where it can rebind hormoneand repeat the whole process of RME. The
`free receptor may also remain sequesteredintracellularly.
`It should be pointed out, however, that not all hormonesdissociate from their
`receptorin the pH 5.5 environment of the endosome [24]. Some hormone-receptor
`complexes require much lower pH valuesfor dissociation to occur. Although not a
`peptide hormone, the iron-transport protein transferrin is a peculiar example of this
`phenomenonandshould be pointed out. In this case, at the neutral pH of the ex-
`tracellular fluid transferrin containing bound iron bindstoits cell surface receptor
`andis internalized. In the low pH environmentof the endosome, iron becomesdis-
`sociated from transferrin, but transferrin remains boundto its receptor. The trans-
`ferrin receptor, with boundtransferrin, is then recycled to the cell surface. With
`iron no longer boundto the transferrin, the transferrin readily dissociates from its
`receptorat 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 documentednotto 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 RMEeventually leads to a decrease
`in the numberof receptors on the cell surface, a phenomenoncalled down-regu-
`lation.
`The distinction between a given receptor being recycled versus degradedis not
`alwaysan 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 accountis that some receptors may be
`spared degradation, but they may not be immediately recycled back to thecell sur-
`face (i.e., they may be sequestered intracellularly). These possible routes of recep-
`tor disappearance and appearanceonthecell surface are summarized schematically
`in Fig. 2.
`Whethera given hormonereceptoris recycled or not during RME dependsnot
`only upon which hormonethe receptor binds, but also upon the cell type and stage
`of differentiation of a given cell. Thus, the insulin receptor has been shownto be
`recycled during RMEinrat adipocytes [28,29], but not in lymphocytes [30]; andit
`is down-regulated in the adult rat liver [31], but not in the fetal rat liver [31].
`
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`137
`
`
`
` recycling
`
`
`internalization —» sequestration
`
`(intracellular pool)
`
`
`degradation
`synthesis
`
`Fig. 2. Possible routes of receptor appearance and disappearance from thecell surface.
`-afl
`
`3. Methods usedto assess receptor-mediated endocytosis
`
`3.1. Morphological approaches
`
`Onalight microscopic level it is possible to visualize the binding of fluorescently
`labelled hormonesto intact cells or to visualize the native hormone with fluorescent
`antibodies [32-35]. Using fluorescently labelled hormones, investigators have ob-
`served a bandof fluorescence defining the circumference of each cell when the
`binding of the fluorescently labelled hormoneto the cells was performed under
`conditions whereinternalization was inhibited (such as at 4°C). Whenthecells were
`allowed to bind hormoneat 4°C, washed to remove unbound hormone, and then
`incubated at 37°C to allow the surface-bound hormoneto be internalized, it was
`possible to observe a concentration of the fluorescence into small patches on the
`cell surface and a subsequentincreasein diffuse fluorescence locatedinsidethecell.
`This experimental approach is powerful in that it allows one to visually determine
`whether under different conditions a hormoneis boundto thecell surface oris in-
`ternalized, and therefore it has been widely used. In orderto identify the particular
`organelles with which the internalized hormone becomesassociated, 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 coupledto 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 '*°1)
`and performing autoradiography [8,9,11]. Alternatively, one can bind the unal-
`tered hormoneto 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 approachis generally preferable in that one need not be concerned
`that the electron dense or radiolabelled hormoneis handled by thecell 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
`hormoneis boundto 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].
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`
`Using these approaches, it has been found that when the binding of the hormone
`to the cells is done at 4°C the hormoneis 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 immunologicalstaining, it is possible to identify
`the intracellular compartments with which the hormoneis 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 hormoneis
`associated with the organelle membrane(and thus probably receptor-bound)oris
`free in the lumen [10].
`Typically, a morphometric analysis is performed where a large numberof micro-
`graphstakenat each time point are examined and the numberofgrains offerritin
`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 ofgrains orparticles associated with a given organelle at each time point
`and arrive ata statistically valid conclusion asto the route of the hormone (and/or
`receptor) during RME [9,11,36].
`
`3.2. Biochemical approaches
`
`In order to study the RMEofa peptide hormone biochemically, it is necessary to
`be able to radiolabel the hormoneto a highspecific activity with '*5I, while retain-
`ing the normal binding and biological properties of the hormone. As discussed in
`the introduction, if one binds the iodinated hormoneto intact cells at 37°C and de-
`tects ligand degradation products in the medium, thatis an indication that RME of
`the hormone maybe occurring. Ligand degradation can be ascertained by analyz-
`ing the molecularsize of the radioactive products by gelfiltration 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 appearanceof acid-soluble radioactivity with metabolic inhibitors (such as NaN;)
`or with compoundsthat inhibit the delivery of the hormoneto the lysosomesorin-
`hibit lysosomalfunction (such as leupeptin, NH,Cl, chloroquine, or monensin; see
`Refs. 38,39).
`When one measures the amount of hormone boundto anintact cell at 37°C, this
`represents a sum of surface-bound hormoneplus hormonethat hassince been in-
`ternalized (andis in an intact or partially degraded form). It should be noted that
`
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`
`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 hormonescan bedissociated from their recep-
`tor under conditions of low pH (pH 3-4) andthus this has been a commonly used
`method [6,36]. An advantage of this methodis that it is a mild treatment and thus
`in some cases one can treat the cells with acid to remove the surface-bound hor-
`moneand then rebind fresh hormone and observea cellular résponse [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
`damagethe receptor and thus cannotbe used if one wishes to subsequently rebind
`fresh hormoneto the cells. Lastly, a variety of other methodstailored 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 doneby 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 releasesall
`the cell-associated radioactivity. Thus, by measuring total cell-associated radioac-
`tivity in one set of cells and releasable radioactivity in anotherset of cells, one can
`calculate the amount of internalized hormone by subtraction. Therefore, one can
`in fact measure hormonebinding to anintact 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 hormonein 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 hormonebinding andinternaliza-
`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 whenthe internal-
`ized receptor is not down-regulated. It should be pointed out that the maximal
`amount of hormoneinternalized and/or degraded will vary depending upon the ex-
`tent of receptor recycling. Thus, if the receptors do not recycle, the maximal amount
`of hormoneinternalized and/or degraded should beless than or equal to the num-
`ber of cell surface receptors. Therefore, the amount of hormonethat is processed
`in this case is dictated by the number of hormonereceptors. If the receptors dore-
`cycle, then the maximal amount of hormoneinternalized and/or degraded should
`exceed the numberofcell surface receptors. In this case the cells can theoretically
`degrade all the added hormone, regardless of the number of hormonereceptors.
`From the biochemical approaches discussed thus far, one can conclude that a given
`hormone maybeinternalized by RME. Conclusive evidence for such internaliza-
`tion, however, can only be obtained by concurrent morphological data as described
`
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`A
`
`—_ >Oo
`
`251_HORMONE
`
`surtace-bound
`
`internalized
`
`surface-bound
`
`;
`
`internalized
`/
`a
`See fe ——’
`se
`
`3
`
`degraded
`
`LENGTH OF INCUBATION (hours)
`
`Fig. 3. Distribution of hormone boundtocells during many roundsof endocytosis. Cells are incubated
`with radiolabelled hormoneforincreasing lengthsof time at 37°C. At various time points, hormonethat
`is surface-bound, internalized or degraded and released into the medium is determinedas described in
`the text. Panel A represents a case wherethecell surface receptor becomes down-regulated; Panel B
`represents a case wherethecell surface receptor is not down-regulated, but insteadis 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 methodto calculate the rate of internalization of a hormone
`is to bind the hormoneto intact cells at 4°C (where no internalization should oc-
`cur), wash the cells to remove unbound hormone and then measure the amountof
`surface-bound radioactivity remaining as a function of time after warmingthecells.
`Unlike the experimental approach described above wherethe cells are allowed to
`continuously bind and internalize hormoneat 37°C and thus undergo many rounds
`of RME(seeFig. 3), under these conditionsthe cells are internalizing only the pre-
`bound hormone and thus are undergoing only one round of RME. A schematic
`example ofresults of this kind of experiment is shown in Fig. 4. Typically, one ob-
`serves a loss of surface-boundradioactivity with a concomitantincrease in the levels
`of internalized radioactivity. Since the internalized hormoneis 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 doneitis 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
`hormonein the lysosomes, the rate of hormone degradation andthe rate of release
`of degradation products. Among these processes, the rate of hormone degradation
`appearsto be limiting [36]. The use of the loss of cell surface-bound radioactivity
`as a measure of the rate of hormoneinternalizationis 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 hormonefrom thecell surface receptor [42].
`
`iaaaia
`
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`
`surface-bound
`=
`
`
`
`BOUND %OFINITIAL51_HORMONE
`
`TIME AFTER WARMING (minutes)
`
`Fig. 4. Distribution of hormone boundtocells during one round of endocytosis. Cells are incubated with
`hormoneat 4°C to saturate the cell surface receptor. At ‘=O, the cells are washed to remove unbound
`hormone and warmed to 37°C. At various time points after warming, the hormonethat 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 hormoneis
`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 asthey 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-
`bound hormoneprovided 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 steadystate. 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 experimentis done be-
`fore any degradation products are released into the medium) versus the integral of
`the surface bound radioactivity [42,44,45]. In additionto the rate of internalization,
`the steady-state analysis described by Wiley and Cunningham allowsoneto calcu-
`late many other parameters pertaining to the hormone-receptorinteraction during
`RME. These include the steady-state association constant for the hormone-recep-
`tor complex (a steady state equivalent of the K, calculated by Scatchard analysis),
`the numberofcell surface receptors, the rate of receptor appearanceatthe 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-
`
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`
`mone is down-regulated, one can use this steady-state model to determineif the
`down-regulation is due to an increasein the rate of internalization of occupied ver-
`sus unoccupied receptors or to a decreasein the rate of appearance of receptors on
`the cell surface. Using this approach, both hCG and EGFhave 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 determineis the in-
`tracellular route of the hormone and receptor. As discussed above, one can use
`morphological approaches to address this question. The morphological approachis
`particularly elegant if one has an antibody to the receptor such that one can simul-
`taneously detect both the hormoneandits 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-
`dosomesor lysosomes[24,47,48]. Thus, one can bind radiolabelled hormoneto the
`cells, allow the cells to internalize the hormonefor 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-
`moneis free or receptor-boundbyprecipitation of the internalized radioactivity by
`polyethylene glycol or ammonium sulfate [24,49]. Furthermore, by analyzing the
`internalized radioactivity in the different compartments on SDS-polyacrylamidegels,
`one can assess whether the hormoneis intact or partially degraded [24,48]. Thus,
`one can determine in which compartmentthe hormonedissociates from its receptor
`and in which compartment degradation of the hormone occurs. Using these tools,
`it has been possible to documentthat unlike many other hormones whichdissociate
`from their receptor in the endosome, hCG remains receptor bound. Thus, the hCG-
`receptor complexis delivered to the lysosomeintact, whereupon the complexis dis-
`sociated [24]. Althoughit can only be directly ascertained that the hormoneis then
`degraded(since it is the hormone whichis radiolabelled), it is assumed that delivery
`of the hCG receptor to the lysosomealso 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 andinternalization to occur, but impedetheintracellular route of the
`internalized hormonereceptor. By using an inhibitor of lysosomal enzymes, such
`as leupeptin, one can ‘trap’ undegraded hormone(andpossibly 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
`compoundssuch as monensin and NH,Cl can be usedto raise the pH in intracell-
`ular organelles [39]. Unfortunately, since pH gradients exist in both endosomes and
`lysosomes(and otherintracellular organelles), these compounds may impede any
`one (or many)ofthe stepsin the transit of the hormone andreceptor, and thus one
`must use additional approaches(as outlined above) to determine in which organelle
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`the hormone(or receptor) has been trapped. Thus, although it has been shownthat
`in many cases monensin and NH,Cltrap the ligand-receptor complex in the en-
`dosomes [47,48,51], it has been documented that in murine Leydig tumorcells these
`compoundsallow the delivery of hCG (boundto 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 sequesteredintracellularly, then one shouldbe‘able 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 measurethe 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 removethe unlabelled hormoneprior 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 RMEof the unlabelled hormone were being
`sequesteredintracellularly. Alternatively, if one detected a decrease in the binding
`activity in the intact cell and in the detergent extract, this would indicate that the
`internalized receptor was being degraded (orinactivated).