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Twelve of fourteen surface epitopes of receptor-bound human chorionic gonadotropin (hCG) being antibody-inaccessible suggest an extensive involvement of the long extracellular domain of the hCG receptor
Molecular and Cellular Endocrinology, 82 (1991) 71-79 0 1991 Elsevier Scientific Publishers Ireland, Ltd. 0303-7207/91/$03.50
MOLCEL
71
02630
Twelve of fourteen surface epitopes of receptor-bound human chorionic gonadotropin (hCG) being antibody-inaccessible suggest an extensive involvement of the long extracellular domain of the hCG receptor S. Schwarz
‘, H. Krude
I, G. Wick ’ and P. Berger
2
’ Institute of General and Experimental Pathology, School of Medicine, University of Innsbruck, A-6020 Innsbruck, Austria, and ’ Institute for Biomedical Ageing Research of the Austrian Academy of Sciences, Innsbruck, Austria (Received
Key words: Monoclonal antibody; G protein-coupled Protein-protein interaction
23 May 1991; accepted
receptor;
Glycoprotein
12 July 1991)
hormone;
Glycoprotein
hormone
receptor;
Summary On the surface of the free (receptor-unbound) form of hCG, we have previously identified 14 topographically distinct epitopes (Schwarz et al. (1986) Endocrinology 118, 189-197; Berger et al. (1990) J. Endocrinol. 125, 301-309). Here we report that only two of them, i.e. the (adjacent) /?3 and /?5 epitopes, can be recognized by ‘25iodine-labeled monoclonal antibodies when hCG was specifically bound to the rat testis hCG receptor. The exclusive accessibility of precisely these two surface epitopes indicates that hCG assumes a defined rather than a stochastic orientation in its receptor-bound state. The inaccessibility of 12 of 14 epitopes is consistent with the idea that the 341 residues long extracellular domain of the recently cloned hCG receptor (MacFarland et al. (1989) Science 245, 494-499) is the ligand binding domain. It is proposed that the extracellular domain is folded in a way that a cavity is formed large enough to accommodate hCG. Thereby, a considerable portion of the total surface of hCG is covered, as reflected by the masking of most of its epitopes.
Introduction The members of the glycoprotein hormone family are human chorionic gonadotropin (hCG), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and thyroid-stimulating hormone (TSH). These are of similar molecular mass (M, = 30), share an elipsoid globular shape and are
Address for correspondence: S. Schwarz, Institute of Genera1 and Experimental Pathology, Faculty of Medicine, Universitv of Innsbruck. A-6020 Innsbruck. Austria. Tel. 43 (5121 507.2266; Fax 43 (512) 507-2507. ’
a:/3 heterodimers. Hormone-specific different p subunits combine noncovalently with a single common (Y subunit and thus confer to the complex the ability to recognize with specificity and high affinity its cognate receptor (Pierce and Parsons, 1981; Ryan et al., 1987) as well as to set in motion G, protein coupling (Gilman, 1987) and further signal transduction events. Yet the precise way in which hCG interacts with the LH/hCG receptor is not known. Considering that surface-exposed amino acid residues of hCG must be involved in receptor contacting (Ryan et al., 19871, some of which may be adjacent to, or even (partly) constitute an
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immunological epitope, we have chosen to use a panel of monoclonal antibodies (MCA). With these, altogether 14 distinct epitopes were identified, five on the LYsubunit (cul to cu5), five on the /3 subunit (61 to /35) and four exclusively on the holo hormone (nj31 to ~~84) (Schwarz et al., 1986; Berger et al., 1990). This data was then applied to study the hCG receptor interaction, (i) by preincubating ‘““I-hCG with an excess of each of these MCAs and subsequent monitoring of the receptor binding ability complexes (Schwarz et al., Of “SI-hCG-MCA 1988), and (ii) as described here, by preincubating rat testis membranes with unlabeled hCG and subsequent monitoring with “‘I-MCA which epitope of receptor-bound hCG is still accessible. In other words, we addressed the question which ‘“‘I-MCA is sterically compatible with the receptor, which translates to: which part(s) of the surface of hCG is (are) left unoccupied by the receptor, or, conversely, how is the ligand binding domain of the receptor folded so that a certain number of surface epitopes are masked (representing, both, a certain part and a certain portion of the total surface area of hCG). Materials
and methods
Highly purified hCG was kindly provided by Dr. W.E. Merz (University of Heidelberg, F.R.G.1 and evaluated against CR-125, a reference standard for hCG kindly provided by the National Pituitary Agency (Baltimore, MD, U.S.A.). Preparation of “’ I- labeled hCG (specific radioactivity: = 1500 Ci/mmol, = 75% being receptorbindable) and of testis membranes (from maIe Sprague-Daw~ey rats, 2 months old) was performed similarly as described (Schwarz et al., 1988). All binding assays were performed in a total incubation volume of 1 ml 50 mM Tris Cl (1% bovine serum albumin (BSA)) buffer at 22 o C on a shaking device. Always, 0.25 mg rat testis membrane protein was used. Incubation with “‘1-hCG or hCG lasted 12-16 h, with ““I-MCAs 4 h. Unbound tigands were removed by washing and centrifugation of the membranes (3200 X g for 20 min at 4 ’ C, repeated 3 times). In all assays, membrane-bound radioactivity was measured.
Radioreceptor competition (displacement) assay Membranes were incubated with = 250,000 cpm of 12’I-hCG (= 0.1-0.2 nM) together with increasing concentrations of unlabeled hCG. Data were evaluated by the computer program ALLFIT (DeLean et al., 1972). Radioreceptor association assay Membranes were incubated with = 250,000 (= 0.1-0.2 nM) for various cpm of ““I-hCG lengths of time. Radioreceptor dissociation assuy Membranes were incubated with - 250,OO~ 12’I-hCG was cpm of ““I-hCG for I2 h, unbound removed and washed membranes were resuspended in 1 ml 50 mM Tris * CI/BSA (1%) buffer alone or buffer containing either 10 nM unlabeled hCG or excess of unlabeled MCA, to assess spontaneous, or ligand-induced or (possibly) MCA-induced dissociation of “‘1-hCG, respectively. Reactions were terminated at given intervals by washing the membranes. Receptor-hCG- “‘I-MCA sandwich assay (standard protocol) Rat testis membranes were preincubated with a single dose of 0.25 nM unlabeled hCG, and after washing incubated with each of our 12’1MCAs (0.05 nM) specific for a single epitope, i.e INN-hFSH-73 (cul) (INN for INNsbruck), INNhFSH-8 (cr2), INN-hCG-15 (a3), INN-hFSH-132 ((~4), INN-hFSH-158 (~5); INN-hCG-10 (a/31), INN-hCG-40 (cup2), INN-hCG-45 (a/33), and INN-hCG-26 (a/34); INN-hCG-2 (@I), INNhCG-22 (p21, INN-bLH-1 and INN-hCG-42 @3), INN-hCG-24 (/34X INN-hCG-58 c/35). Details of the preparation, purification, characterization and radiolabeling with ‘2siodine of the MCAs used here were given previously (Kofler et al., 1981; Berger et al., 1984, 1988, 1990; Schwarz et al., 1986, 1988, 1991a). Specific radioactivities were - 1500 Ci/mmol IgG. The very same preparations of “s1-MCAs were used in parallel experiments in which hCG was preincubated with a compatible capture MCA (adsorbed to polystyrene tubes) instead of testis membranes, as described elsewhere (Schwarz et al., 1986, 1991a).
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Sandwich titration protocol Similar as the standard protocol, only that instead of a single dose of hCG increasing concentrations (0.001-l nM) of hCG were used. To assess specificity, in some experiments rat liver instead of testis membranes were used (equal amounts, equally prepared).
Sandwich saturation protocol Similar as the standard protocol, only that instead of a single dose of ‘251-MCA increasing concentrations (0.01-2 nM) of ““I-MCA were used (membranes were first incubated with 0.1 nM hCG). The same 12’I-MCA saturation experiment was performed on solid phase (~4-MCA (INN-hFSH-132)-bound hCG. Data were transformed according to Scatchard (1949) and evaluated by a computer program (Schwarz, 1979).
Sandwich self- and cross-inhibition protocol Similar as the standard protocol, only that the incubation with 12’1-MCA (0.05 nM) was carried out concurrently with increasing concentrations (0.001-10 nM) of unlabeled MCAs against the same or a different epitope. Results As shown in Fig. 1, unlabeled hCG displaced binding of ‘*“I-hCG from rat testis membranes
log [hCG], M Fig. 1. Binding of ‘2’1-hCG (decreasing; closed circles) and of the p3 epitope-specific 12”I-MCA INN-bLH-1 (increasing; open squares) as a function of the concentration of unlabeled hCG. Values are expressed as percent of the respective maximal responses. Note the similarity of the IC,, and ECS,,. Triangles indicate ‘2sI-INN-bLH-l binding to rat liver membranes preincubated with hCG. Each point is the mean of duplicate determinations of a single experiment repeated at least twice.
with an IC,,, of 3.5 k 0.3 (SE, n = 3) X 10-l’ M and a pseudo-Hill coefficient of = 1, indicating high affinity hCG binding to a single class of receptor sites. In kinetic experiments, the 22°C half times of association and dissociation of 12’1hCG to and from rat testis membranes were found to be = 2 and = 48 h, respectively. The t 1/2diss was 48 h even when excess amounts of unlabeled hCG or MCA were present (results not shown). These values are in accordance with those reported on natural (Chen et al., 1979; Pierce and Parson, 1981; Ryan et al., 1987) as well as cloned LH/hCG receptors (McFarland et al., 1989; Xie et al., 1990) and thus underline the functional integrity of the here employed test system (i.e. hCG and receptor). Unlabeled hCG that only marginally (less than 5%) dissociates from the receptor within 4 h should be detectable also indirectly, namely by 12”1-MCA, in particular as an MCA itself does not promote dissociation of 12”1-hCG, as mentioned above. In the sandwich protocol used, aliquots of rat testis membranes were preincubated with a receptor-saturating concentration (2.5 X lO_” M) of hCG, subsequently thoroughly washed to remove unbound hCG and finally exposed to an equal dose of each of our 14 different 12sI-MCAs. As shown by the black bars in Fig. 2, only two of them (i.e. the P-MCA INN-bLH-1 and the /35-MCA INN-hCG-58) could recognize their respective epitopes on receptor-bound hCG; all other MCAs could not. For this negative result, neither insufficient ‘reactivity’ (due to iodination damage) nor insufficient affinity can account: as shown by the stipped bars in Fig. 2, every of the here used preparations of “‘1-MCAs could very well recognize hCG that was bound to a compatible capture MCA. And, the affinity of all MCAs (except three, i.e. al, ap3, pl) for free (i.e. receptor-unbound, soluble) hCG is equal or even up to 30 times higher than that of INNbLH-1 (Berger et al., 1984, 1990; Schwarz et al., 1991a) (see bottom row of Fig. 2). In order to assess dose dependency and specificity of ‘251-MCA binding, sandwich titration experiments were performed. As shown in Fig. 1 (open squares), a constant amount of ‘2sI-INNbLH-1 added produced a response proportional to the dose of hCG present. This response was
74
saturable (= 3 x IO-"'M) and was half maximal (EC,,, = 2.5 X lo-" M) at a concentration of hCG similar to its IC,,, in displacing ‘2”I-hCG. A dose of 2.5 x lo-“’ M of hCG thus being shown to be indeed receptor saturating excludes the possibility that insufficient receptor occupancy by hCG could account for the unreactivity of some or all of the 12 negative ““I-MCAs shown in Fig. 2. Besides this, the shape and the EC,,, of this response clearly indicate that I25I-MCA binding to hCG is specific whereby hCG itself is specifically bound to rat testis hCG receptors: ‘2SI-MCA binding in the absence of hCG was equal to that seen with rat liver membranes preincubated with hCG (Fig. I>. Further, a sandwich saturation protocol was employed in order to determine the affinity of an MCA for receptor-bound hCG. Rat testis membranes, preincubated with hCG, were exposed to increasing concentrations of ‘*“I-INN-bLH-1 as well as, in parallel, 1’SI-INN-hCG-42 (a sister-
Ka(MCAs): 0.1 33 nd 3 Ka(receptor): 286
1
1 15 0.1 2 0.3 11 1
1
3
Fig. 2. Epitope accessibility pattern of receptor-bound hCG (black bars) as probed by the receptor-hCG-““I-MC/\ sandwich approach using 14 different MCAs against 14 topographically distinct surface epitopes of hCG. As a positive control, i.e. for assessing the principal reactivity, the same preparation of each ‘z51-MCA was tested in parallel also on hCG bound to a capture MCA (stipped bars) of known compatibility (Schwarz et al., 1986, 1991; Berger et al., 1990); note that for most ‘2s1-MCAs responses of specific binding were greater than 15,000 cpm. The equilibrium affinity (K,, I.mol-’ X 10’) of each MCA for free hCG, determined at 4 ’ C by displacement with unlabeled hCG, is given underneath (rounded values taken from Berger et al., 1990; Schwarz et al.. 1991). For comparison, also the K,, of the rat testis hCG receptor for hCG is given (taken from Fig. 1). Background of iZf-MCA binding was assessed by omission of hCG, or by using equal amounts of rat liver instead of rat testis membranes. Each bar is the mean of duplicate determinations of a single experiment repeated twice.
O,i)2
3
50
100
lz51-MCA bound (fmole/mg protein)
a4 MCA.b~und hCG
0,oO 0
5 10 125 I-MCA bound (x 10 .‘* mole/L)
Fig. 3. Scatchard plot representations of two different MCAs specific for the /33 epitope of hCG, as obtained at 22°C in “‘I-MCA sandwich saturation experiments using either receptor-bound hCG (total incubation volume: 1 ml; left panel) or cu4 capture MCA-bound hCG (total incubation volume: 0.2 ml; right panel). Note that both /33-MCAs (i.e. INN-bLH-I, INN-hCG-42) recognize the same number of epitopes, i.e. 100 fmol/mg protein (=0.018 nM) (left panel). This B,,>,* concords with the number of hCG molecules bound, as determined in a saturation assay using ‘a51-hCG (not shown). Note further that the affinity of INN-bLH-1 is 9 times higher for recept~~r-bound hCG than for (~4 MCA-bound hCG. Each point in these plots is the mean of duplicate determinations of a single experiment repeated twice.
MCA also directed towards the /33 epitope: Berger et al., 1990). Scatchard plots shown in Fig. 3 (left panel) indicate that the affinity (I(,) of INN-bLH-1 is 91 k 8 x lo8 1. mol-‘, a number that is = 10 or = 90 times higher than the K,,of INN-bLH-1 for cy4-MCA-bound hCG (Fig. 3, right panel) or soluble hCG (i.e. = 1 X 10’ I * mol_ ‘, Fig. 2), respectively. INN-hCG-42, whose affinity for receptor-bound hCG was found to be = 16 times lower than that of INN-bLH-1 (K;$ = 5.6It0.2 X 10’ 1. mol-‘1 (Fig. 3, left panel), was still of sufficient strength as to recognize the same number of p3 epitopes on receptor-bound hCG as did INN-bLH-1. The J33 and /35 epitopes are adjacent on receptor-unbound hCG (Berger et al., 1990), because binding of the respective MCAs is mutually exclusive (see compatibility table shown elsewhere: Schwarz et al., 1991). To test whether this applies also for receptor-bound hCG, a self- and cross-inhibition experiment was performed. As shown in Fig. 4, ‘2’I-INN-bLH-1 (p3) binding to receptor-bound hCG could dose-dependently be
log [MCA], M Fig. 4. Inhibition of binding of the p3 epitope-specific “‘IMCA INN-bLI-I-I by increasing doses of unlabeled INN-bLH1 or INN-hCG-58, as determined in a sandwich self- and cross-inhibition experiment. Note that the MCA specific for the 83 epitop~-adjacent g5 epitope is incompatible with ‘2”I-MCA INN-bLl_S-1just as is INN-b~H-1 itself. Blank (zero point) was determined in hCG-preincubated membranes which were subsequent to washing exposed to ‘251-INN-bLH-l in the absence of INN-bLH-1 (maximal binding of “‘I-INNbLH-I). Each point is the mean of duplicate determinations of a single experiment repeated twice.
inhibited by unlabeled INN-hCG-58 (/35) and, as positive control, also by itself. Note that the reciprocal of the IC,, (1.5 x lo-” M) of INN-bLH1, i.e 67 X lo8 1. mol-‘, is comparable to its K, = 91 X 10s 1. mol-’ determined by saturation assay (Fig. 3, left panel). Discussion Based on our previous compatibility assessment using paired MCA binding (capture MCAhCG-‘25f-MCA sandwich), 14 epitopes were distinguished which are distributed in a characteristic topographical manner on the surface of hCG (Schwarz et al., 1986; Berger et al., 19901. Considering the known diameter of an antibody’s Fab cylinder on one hand (Novotny et al., 1983) and the Stokes radius of hCG on the other (Pierce and Parson, 1981), these 14 epitopes are very likely spread over a considerable portion of the water accessible surface of hCG rather than clustered in a small district (Schwarz et al., 1986). Hence, they can be utilized as coordinates or landmarks for testing hCG in its receptor-bound state. As shown in Fig. 2, 12 of 14 epitopes were invisible on receptor-bound hCG. Since incom-
plete receptor occupancy, insufficient MCA affinity and/or ‘reactivity’ could be ruled out by appropriate experiments, as discussed already above, only two factors remain to be considered as possible causes for the ‘loss’ of epitopes: (i) receptorinduced conformational change of hCG, and (ii) steric hindrance by the receptor. Despite hoio-hCG is structurally stabilized by a great number of disulfide bonds within its subunits (Pierce and Parsons, 1981), a change of hCG’s conformation within certain limits is still possible, such as following subunit association (Strickland and Puett, lYSZ), or deglycosylation, or, for that case, receptor binding. Consistent with obse~ations obtained by others indicating that such changes can be ‘sensed’ by antibodies (Keutmann et al., 1985; Norman et al., 1985; Hojo and Ryan, 1987; Moyle et al., 1987), we have reported on 2- to = 90-fold differences in MCA affinity for holo-hCG vs. the subunits (Berger et al., 19901, for native vs. deglycosylated hCG (Schwarz et al., 1991a1, and, as shown here (Fig. 31, for for vs. capture MCA-bound hCG or for free vs. receptor-bound hCG. We have also addressed the possibility that a conformational change could have led to ‘dislocation’ of epitopes, i.e. a change in the relative distances of two epitopes, resulting in change of the mutual compatibility of a pair of respective MCAs. Yet, alterations in the epitope topography could neither be detected between native and deglycosylated hCG (Schwarz et al., 1991a) nor between free and receptor-bound hCG (Fig. 4). Thus, ‘natural’ changes in the overall conformation can ‘reshape’ every or certain of hCG’s surface epitopes, leading to increase (e.g. INN-bLH-1, /33 epitope) but also decrease of MCA affinity (e.g. INN-hCG-58, p5 epitope), similar to what was observed by Moyle et al. (1987). However, unlike denaturation, such changes do not appear to be profound enough as to disrupt the antigenic topography or to destroy the antigenic integrity with consequent unreactiveness of MCAs. Thus, epitopes appear to be fairly robust landmarks of a globular protein’s surface. Otherwise, the intactness of the /33 and /35 epitopes would be extremely difficult to reconcile with the assumption that 12 of 14 epitopes could have been destroyed.
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The experiment shown in Fig. 3 (left panel) indicates that INN-hCG-42, in spite of even 16fold lower affinity for receptor-bound hCG, could just as reliably detect the p3 epitope as its sister MCA INN-bLH-1. In addition, it is shown that every of the 14 different ‘2”1-MCAs could very we11 recognize hCG when bound to a compatible capture MCA (stippled bars in Fig. 21, whether being of high or low affinity (K, values ranging from 33 to 0.1 X 10’ 1 *moll’l. Thus, in any sequential sandwich protocol (be it receptor-hCG‘251-MCA or capture MCA-hCG-““I-MCA), the affinity of the detection MCA determines the degree of ““I-MCA binding but plays otherwise no decisive role, i.e. whether binding is possible at all or not. The reason is that in each of the incubations only two reaction partners are present, i.e. receptor and hCG in the first step and receptor-bound hCG and “‘1-MCA in the second. In addition, receptor-bound hCG does not dissociate and therefore not partition, so that also the relative affinity of the ““I-MCA is of little importance (INN-bLH-1 was reactive in spite of = 300 times lower K, compared to that of the receptor, see Fig. 2). Thus, the advantage of the sequential sandwich approach over protocols employing concurrent incubation of membranes with hCG and 12’1-MCA (or with ‘Z”I-hCG and MCA) is that it provides a simple all-or-none type end point, that is a response which is solely dependent on the compatibility between the two binding reagents (receptor and “‘I-MCA), i.e. freedom from steric hindrance exerted by the respective other reagent. This has previously clearly been shown in the capture MCA-hCG- “$1-MCA configuration where reciprocal combinations of two compatible MCAs yielded always identical results (Schwarz et al., 1986). Thus, the inaccessibility of 12 of 14 epitopes, as shown in Fig. 2, is most likely due to masking by moieties of the receptor. The LH/hCG receptor (McFarland et al., 1989) as well as the receptors for TSH (Parmentier et al., 1989) and FSH (Sprengel et al., 1990) have recently been cloned. Their EDNA-derived primary structures indicate that they share architecture and membrane topology with the G protein-coupled family of receptors (prototype being rhodopsin). However, unlike members that bind small ligands such as
adrenoceptors, the hCG receptor features in addition a long (341 residues) extracellular domain (consisting of 14 tandemly arranged leucine-rich repetitive motifs, presumably archetypic modules of protein-protein interaction). The seven transmembrane helices of all G protein-coupled receptors have been shown to be ring-like arranged (Frielle et al., 19881, thus f$rming a pocket whose inner diameter is = 15 A, thus Dable to fully accommodate a ligand of = 10 A length (e.g. epinephrinel (Strader et al., 1989) but, very probably, not a ligand of a Stokes radius of = 37 A (Pierce and Parsons, 1981) such as hCG. Hence, the only candidate moiety to bind hCG appears to be that long extracellular domain. Antibody labeling and site directed mutagenesis experiments have only recently verified that this domain is truly extracellular (Rodriguez and Segaloff, 1990) and that it binds hCG with an affinity comparable to the non-truncated hCG receptor (Xie et al., 1990; compare also Wadsworth et al., 1990 as to the TSH receptor). However, it is still not understood how and how much of the extracellular domain participates in forming the actual contact(s) with hCG: is it only a short stretch of the N-terminal portion or are muhiple stretches required that are themselves continuous or discontinuous? Peptide competition experiments have suggested the involvement of contact sequences spread over the entire length of this domain on one hand (especially residues 22-35) (Roche and Kitzmann, 1990) and of the hCG molecule on the other ((~1-15, n2646, ~~75-92, p38-57, /?93- 1001(Ryan et al., 19137). The question is, how is the extracehular domain folded to allow such a multifacetted interaction? The results of the here presented epitope accessibility paradigm may provide clues to such questions. The total area of all epitopes combined represents a certain fraction of and is thus representative for the total surface area of hCG. Hence, the fact that most (i.e. 12 of 14) epitopes were inaccessible suggests that moieties of the hCG receptor cover a considerabIe portion of the surface of hCG, relatively more of its cy than j3 subunit, as was suggested also by others (Ji and Ji, 1981; Moyle et al., 1982; Milius et al., 1983; Hwang and Menon, 1984). So much of hCG concealed seems difficult to reconcile with a com-
77
pactly folded superstructure of the extracellular domain (model A of Frazier et al., 1990). For the same but yet another reason also model B, implying immersion of the hormone into the transmembrane pocket, seems unlikely: given that the extracellular loops of the hCG receptor are not longer than those of the adrenoceptors, also the transmembrane pocket is likely to be of similar volume, that is, too small as to accommodate hCG.
Lp3,
We therefore propose a tentative model (schematically portrayed in Fig. 5) in which the extracellular domain is viewed as a rather open and flexible structure that reaches out into the extracellular space. As such it may first capture with its N-terminal part (e.g. residues 22-35 and some others) a certain part of hCG. In an adaptive, ‘hand-and-glove’-like mode of interaction, a sequence of multiple further discrete proteinprotein contacts is then established, as two obser-
ps epitopes accessible
Fig. 5. Schematic and tentative model of the differential modes of interaction of epinephrine (to the right) and of hCG (to the left) with their respective receptors representing prototypes of the neuronal and the endocrine class of G protein-coupled receptors, respectively. The putative structures of these receptors were taken from McFarland et al. (19891, Frielle et al. (1988) and Strader et al. (19891, and modified for incorporating the data obtained here by the epitope accessibility paradigm. hCG is depicted as a compact globular protein carrying I4 surface epitopes some of which being partly or fully on the back (i.e. tu3, cu5, @2, ~$41. The long extracellular domain of the hCG receptor is shown as an extended structure which in a ‘hand-and-glove’-like manner binds with high affinity and long occupancy hCG and thereby extensively covers the surface of hCG, i.e. masks most of its epitopes (for clarity, only accessible epitopes being shown; dark areas). This is in contrast to a ‘key-and-lock’-type, low affinity and short occupancy interaction of epinephrine and similar neurotransmitters with the transmembrane pocket of neuronal-type receptors. In both types, the third intracellular loop couples in an agonist-dependent manner with the a subunit of the G protein ((~/3y), thereby promoting (spy dissociation and subsequent activation of the adenylyl cyclase (AC) (Gilman, 1987). NANA, N-acetylneuraminic acid.
7x
vations suggest: (i) the relatively slow association kinetics (t,,2ass = 2 h), and (ii) the obvious perturbation sensitivity of this process: preincubation of 12”I-hCG with MCA abrogated the receptor-binding ability of ‘2SI-hCG-MCA complexes, no matter what part (epitope) of the surface of hCG was occupied (even the /33-MCA did so), as reported previously (Schwarz et al., 1988). At the end of this binding process, most of the epitopes of hCG are (passively) concealed by the ligand-binding moietv of the rece&or in which hCG has assumed a defined orientation. For, would the binding behavior be stochastic, many different orientations would be permitted, each with a different epitope accessibility pattern, so that, globally, all 14 epitopes would be accessible. Whether certain residues of either the hormone or the hormone-occupied extracellular domain (receptor autorecognition), interact in addition with the extracellular loops of the receptor, awaits further studies. This epitope accessibility paradigm has also been applied to the antagonist deglycosylated hCG in order to elucidate the role of the carbohydrate moieties on receptor interaction and signal transduction (Schwarz et al., 1991b). The observation that the p3 epitope was accessible on receptor-bound hCG may open important diagnostic and therapeutic possibilities, such as the use of this MCA as a means to (scintigraphically) visualize the degree of LH/ hCG receptor occupancy in vivo, or as a vehicle to specifically target a cytotoxic agent to a Leydig cell tumor expressing hCG receptors. Acknowledgements
This work was supported in part by the Austrian Academy of Sciences. We thank Ms. Regine Gerth, Renate Goerz, Irene Gaggl and Barbara Mayer for their skillful technical assistance and the National Pituitary Agency in Baltimore, MD, U.S.A., and Dr. W.E. Merz (University of Heidelberg, F.R.G.) for having kindIy supplied us with various glycoprotein hormone preparations. References Berger, P., Kofler, R. and Wick, Immunol. 5, 157-161.
G. (1984) Am. J. Reprod.
.
Berger, P., Panmoung, W., Khashabi, D., Mayregger, B. and Wick, G. (1988) Endocrinology 123, 2351-2359. Berger, P., Klieber, R., Panmoung, W.. Madersbacher, S., Wolf, H. and Wick, G. (1990) .I. Endocrinol. 125, 301-309. Chen, C.J.H., Lindeman, J.G., Trowbridge, C.T. and Bhalla, V.K. (1979) Biochim. Biophys. Acta 5X4.407-435.436-453. De Lean, A., Munson, P.J.‘&l Rodbard, D. (1972) Am. J. Physiol. 235, E97-E102. Frazier, A.L., Robbins, L.S., Stork, P.J., Sprengel, R., Segaloff, D.L. and Cone, R.D. (1990) Mol. Endocrinol. 4, 12641276. Frielle, L.T., Daniel, K.W., Caron, M.G. and Lefkowitz, R.J. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 9494-0498. Gilman, A.G. (1987) Annu. Rev. Biochem. 56, 615-649. Hojo, H. and Ryan, R.J. (198.5) Endocrinology 117.2428-2434. Hwang, J. and Menon, K.M.J. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 4667-4671. Ji, 1. and Ji, T.H. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 5465-5469. Keutmann, H.T., Johnson, L. and Ryan, R.J. (1985) FEBS Lett. 185, 333-339. Kofler, R., Kalchschmid, E., Berger, P. and Wick, G. (1981) Immunobiology 160, 196-207. McFarland, K.C., Sprengel, R., Phillips. H.S., Kiihler, M., Rosemblit, N., Nickolics, K., Scgaloff. D.L. and Seeburg. P.H. (1989) Science 245. 494-499. Milius, R.P., Midgley, Jr., A.R. and Birken. S. (1983) Proc. Natl. Acad. Sci. U.S.A. 80. 7375-7379. Moyle, W.R., Ehrlich, P.H. and Canfield, R.E. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2245-2249. Moyle, W.R., Pressey, A., Dean-Emig, D., Anderson, D.M., Demeter, M., Lustbader, J. and Ehrlich, P.H. (1987) J. Biol. Chem. 262, 16920-16926. Normal, R.J., Poulton. T., Gard, T. and Chard, T. (1985) J. Clin. Endocrinol. Metab. 61, 11)31-1038. Novotny, J., Bruccoleri, R.E., Newell, J., Murphy, D.. Haber, E. and Karplus, M. (1983) J. Biol. Chem. 258, 14433-14437. Parmentier. M., Libert, F., Meanhaut, C., Lefort, A.. Gerard, C., Perret, J., Van Sande, J., Dumont. J.E. and Vassart. G. (1989) Science 246, 1620-1622. Pierce, J.G. and Parsons, T.G. (1981) Annu. Rev. Biochem. 50, 465-495. Roche, P.C. and Kitzmann. K.A. (1990) 72nd Annual Meeting of The Endocrine Society, Atlanta, GA, June 20-23, 1990. Abstract 649. Rodriguez, M.C. and Segaloff, D.L. (1990) Endocrinology 127, 674-681. Ryan, R.J., Keutmann, H.T., Charlesworth, M.C., McCormick, D.J., Milius, R.P., Calvo. F.O. and Vutyavanich, T. (1987) Recent Progr. Horm. Res. 43, 383-429. Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 51, 660-672. Schwarz. S. (1979) J. Steroid Biochem. 1 I, 1641-1649. Schwarz, S., Berger, P. and Wick, G. (19%) Endocrinology 118, 189-197. Schwarz. S., Berger, P., Nelboeck, E., Kashabi, D., Panmoung, W., Klieber, R. and Wick, G. (1988) J. Receptor Res. 8, 437.-453. Schwarz, S., Krude, H., Klieber, R.. Dirnhofer, S., Lotters-
79 berger, C., Merz, WE.. Wick, G. and Berger, P. (1991a) Mol. Cell. Endocrinol. 80, 33-40. Schwarz, S., Krude, H., Merz, W.E., Lottersberger, C., Wick, G. and Berger, P. (1991b) Biochem. Biophys. Res. Commun. (in press). Sprengel, R., Braun, T., Nikohcs, K., Segaloff, D.L. and Seeburg, P.H. (1990) Mol. Endocrinol. 4, 525-530. Strader, CD., Sigal, I.S. and Dixon, R.F. (1989) FASEB J. 3, 1825- 1832.
Strickland, J.W. and Puett, D. 11982) J. Biol. Chem. 257, 2954-2960. Wadsworth, H.L., Chazenbalk, G.D., Nagayama, Y., Russo, D. and Rapoport, B. (1990) Science 249. 1423-1425. Xie, Y.B., Wang, H.Y. and Segaloff, D.L. (1990) J. Biol. Chem. 26.5, 21411-21414.
MOLCEL
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02630
Twelve of fourteen surface epitopes of receptor-bound human chorionic gonadotropin (hCG) being antibody-inaccessible suggest an extensive involvement of the long extracellular domain of the hCG receptor S. Schwarz
‘, H. Krude
I, G. Wick ’ and P. Berger
2
’ Institute of General and Experimental Pathology, School of Medicine, University of Innsbruck, A-6020 Innsbruck, Austria, and ’ Institute for Biomedical Ageing Research of the Austrian Academy of Sciences, Innsbruck, Austria (Received
Key words: Monoclonal antibody; G protein-coupled Protein-protein interaction
23 May 1991; accepted
receptor;
Glycoprotein
12 July 1991)
hormone;
Glycoprotein
hormone
receptor;
Summary On the surface of the free (receptor-unbound) form of hCG, we have previously identified 14 topographically distinct epitopes (Schwarz et al. (1986) Endocrinology 118, 189-197; Berger et al. (1990) J. Endocrinol. 125, 301-309). Here we report that only two of them, i.e. the (adjacent) /?3 and /?5 epitopes, can be recognized by ‘25iodine-labeled monoclonal antibodies when hCG was specifically bound to the rat testis hCG receptor. The exclusive accessibility of precisely these two surface epitopes indicates that hCG assumes a defined rather than a stochastic orientation in its receptor-bound state. The inaccessibility of 12 of 14 epitopes is consistent with the idea that the 341 residues long extracellular domain of the recently cloned hCG receptor (MacFarland et al. (1989) Science 245, 494-499) is the ligand binding domain. It is proposed that the extracellular domain is folded in a way that a cavity is formed large enough to accommodate hCG. Thereby, a considerable portion of the total surface of hCG is covered, as reflected by the masking of most of its epitopes.
Introduction The members of the glycoprotein hormone family are human chorionic gonadotropin (hCG), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and thyroid-stimulating hormone (TSH). These are of similar molecular mass (M, = 30), share an elipsoid globular shape and are
Address for correspondence: S. Schwarz, Institute of Genera1 and Experimental Pathology, Faculty of Medicine, Universitv of Innsbruck. A-6020 Innsbruck. Austria. Tel. 43 (5121 507.2266; Fax 43 (512) 507-2507. ’
a:/3 heterodimers. Hormone-specific different p subunits combine noncovalently with a single common (Y subunit and thus confer to the complex the ability to recognize with specificity and high affinity its cognate receptor (Pierce and Parsons, 1981; Ryan et al., 1987) as well as to set in motion G, protein coupling (Gilman, 1987) and further signal transduction events. Yet the precise way in which hCG interacts with the LH/hCG receptor is not known. Considering that surface-exposed amino acid residues of hCG must be involved in receptor contacting (Ryan et al., 19871, some of which may be adjacent to, or even (partly) constitute an
72
immunological epitope, we have chosen to use a panel of monoclonal antibodies (MCA). With these, altogether 14 distinct epitopes were identified, five on the LYsubunit (cul to cu5), five on the /3 subunit (61 to /35) and four exclusively on the holo hormone (nj31 to ~~84) (Schwarz et al., 1986; Berger et al., 1990). This data was then applied to study the hCG receptor interaction, (i) by preincubating ‘““I-hCG with an excess of each of these MCAs and subsequent monitoring of the receptor binding ability complexes (Schwarz et al., Of “SI-hCG-MCA 1988), and (ii) as described here, by preincubating rat testis membranes with unlabeled hCG and subsequent monitoring with “‘I-MCA which epitope of receptor-bound hCG is still accessible. In other words, we addressed the question which ‘“‘I-MCA is sterically compatible with the receptor, which translates to: which part(s) of the surface of hCG is (are) left unoccupied by the receptor, or, conversely, how is the ligand binding domain of the receptor folded so that a certain number of surface epitopes are masked (representing, both, a certain part and a certain portion of the total surface area of hCG). Materials
and methods
Highly purified hCG was kindly provided by Dr. W.E. Merz (University of Heidelberg, F.R.G.1 and evaluated against CR-125, a reference standard for hCG kindly provided by the National Pituitary Agency (Baltimore, MD, U.S.A.). Preparation of “’ I- labeled hCG (specific radioactivity: = 1500 Ci/mmol, = 75% being receptorbindable) and of testis membranes (from maIe Sprague-Daw~ey rats, 2 months old) was performed similarly as described (Schwarz et al., 1988). All binding assays were performed in a total incubation volume of 1 ml 50 mM Tris Cl (1% bovine serum albumin (BSA)) buffer at 22 o C on a shaking device. Always, 0.25 mg rat testis membrane protein was used. Incubation with “‘1-hCG or hCG lasted 12-16 h, with ““I-MCAs 4 h. Unbound tigands were removed by washing and centrifugation of the membranes (3200 X g for 20 min at 4 ’ C, repeated 3 times). In all assays, membrane-bound radioactivity was measured.
Radioreceptor competition (displacement) assay Membranes were incubated with = 250,000 cpm of 12’I-hCG (= 0.1-0.2 nM) together with increasing concentrations of unlabeled hCG. Data were evaluated by the computer program ALLFIT (DeLean et al., 1972). Radioreceptor association assay Membranes were incubated with = 250,000 (= 0.1-0.2 nM) for various cpm of ““I-hCG lengths of time. Radioreceptor dissociation assuy Membranes were incubated with - 250,OO~ 12’I-hCG was cpm of ““I-hCG for I2 h, unbound removed and washed membranes were resuspended in 1 ml 50 mM Tris * CI/BSA (1%) buffer alone or buffer containing either 10 nM unlabeled hCG or excess of unlabeled MCA, to assess spontaneous, or ligand-induced or (possibly) MCA-induced dissociation of “‘1-hCG, respectively. Reactions were terminated at given intervals by washing the membranes. Receptor-hCG- “‘I-MCA sandwich assay (standard protocol) Rat testis membranes were preincubated with a single dose of 0.25 nM unlabeled hCG, and after washing incubated with each of our 12’1MCAs (0.05 nM) specific for a single epitope, i.e INN-hFSH-73 (cul) (INN for INNsbruck), INNhFSH-8 (cr2), INN-hCG-15 (a3), INN-hFSH-132 ((~4), INN-hFSH-158 (~5); INN-hCG-10 (a/31), INN-hCG-40 (cup2), INN-hCG-45 (a/33), and INN-hCG-26 (a/34); INN-hCG-2 (@I), INNhCG-22 (p21, INN-bLH-1 and INN-hCG-42 @3), INN-hCG-24 (/34X INN-hCG-58 c/35). Details of the preparation, purification, characterization and radiolabeling with ‘2siodine of the MCAs used here were given previously (Kofler et al., 1981; Berger et al., 1984, 1988, 1990; Schwarz et al., 1986, 1988, 1991a). Specific radioactivities were - 1500 Ci/mmol IgG. The very same preparations of “s1-MCAs were used in parallel experiments in which hCG was preincubated with a compatible capture MCA (adsorbed to polystyrene tubes) instead of testis membranes, as described elsewhere (Schwarz et al., 1986, 1991a).
73
Sandwich titration protocol Similar as the standard protocol, only that instead of a single dose of hCG increasing concentrations (0.001-l nM) of hCG were used. To assess specificity, in some experiments rat liver instead of testis membranes were used (equal amounts, equally prepared).
Sandwich saturation protocol Similar as the standard protocol, only that instead of a single dose of ‘251-MCA increasing concentrations (0.01-2 nM) of ““I-MCA were used (membranes were first incubated with 0.1 nM hCG). The same 12’I-MCA saturation experiment was performed on solid phase (~4-MCA (INN-hFSH-132)-bound hCG. Data were transformed according to Scatchard (1949) and evaluated by a computer program (Schwarz, 1979).
Sandwich self- and cross-inhibition protocol Similar as the standard protocol, only that the incubation with 12’1-MCA (0.05 nM) was carried out concurrently with increasing concentrations (0.001-10 nM) of unlabeled MCAs against the same or a different epitope. Results As shown in Fig. 1, unlabeled hCG displaced binding of ‘*“I-hCG from rat testis membranes
log [hCG], M Fig. 1. Binding of ‘2’1-hCG (decreasing; closed circles) and of the p3 epitope-specific 12”I-MCA INN-bLH-1 (increasing; open squares) as a function of the concentration of unlabeled hCG. Values are expressed as percent of the respective maximal responses. Note the similarity of the IC,, and ECS,,. Triangles indicate ‘2sI-INN-bLH-l binding to rat liver membranes preincubated with hCG. Each point is the mean of duplicate determinations of a single experiment repeated at least twice.
with an IC,,, of 3.5 k 0.3 (SE, n = 3) X 10-l’ M and a pseudo-Hill coefficient of = 1, indicating high affinity hCG binding to a single class of receptor sites. In kinetic experiments, the 22°C half times of association and dissociation of 12’1hCG to and from rat testis membranes were found to be = 2 and = 48 h, respectively. The t 1/2diss was 48 h even when excess amounts of unlabeled hCG or MCA were present (results not shown). These values are in accordance with those reported on natural (Chen et al., 1979; Pierce and Parson, 1981; Ryan et al., 1987) as well as cloned LH/hCG receptors (McFarland et al., 1989; Xie et al., 1990) and thus underline the functional integrity of the here employed test system (i.e. hCG and receptor). Unlabeled hCG that only marginally (less than 5%) dissociates from the receptor within 4 h should be detectable also indirectly, namely by 12”1-MCA, in particular as an MCA itself does not promote dissociation of 12”1-hCG, as mentioned above. In the sandwich protocol used, aliquots of rat testis membranes were preincubated with a receptor-saturating concentration (2.5 X lO_” M) of hCG, subsequently thoroughly washed to remove unbound hCG and finally exposed to an equal dose of each of our 14 different 12sI-MCAs. As shown by the black bars in Fig. 2, only two of them (i.e. the P-MCA INN-bLH-1 and the /35-MCA INN-hCG-58) could recognize their respective epitopes on receptor-bound hCG; all other MCAs could not. For this negative result, neither insufficient ‘reactivity’ (due to iodination damage) nor insufficient affinity can account: as shown by the stipped bars in Fig. 2, every of the here used preparations of “‘1-MCAs could very well recognize hCG that was bound to a compatible capture MCA. And, the affinity of all MCAs (except three, i.e. al, ap3, pl) for free (i.e. receptor-unbound, soluble) hCG is equal or even up to 30 times higher than that of INNbLH-1 (Berger et al., 1984, 1990; Schwarz et al., 1991a) (see bottom row of Fig. 2). In order to assess dose dependency and specificity of ‘251-MCA binding, sandwich titration experiments were performed. As shown in Fig. 1 (open squares), a constant amount of ‘2sI-INNbLH-1 added produced a response proportional to the dose of hCG present. This response was
74
saturable (= 3 x IO-"'M) and was half maximal (EC,,, = 2.5 X lo-" M) at a concentration of hCG similar to its IC,,, in displacing ‘2”I-hCG. A dose of 2.5 x lo-“’ M of hCG thus being shown to be indeed receptor saturating excludes the possibility that insufficient receptor occupancy by hCG could account for the unreactivity of some or all of the 12 negative ““I-MCAs shown in Fig. 2. Besides this, the shape and the EC,,, of this response clearly indicate that I25I-MCA binding to hCG is specific whereby hCG itself is specifically bound to rat testis hCG receptors: ‘2SI-MCA binding in the absence of hCG was equal to that seen with rat liver membranes preincubated with hCG (Fig. I>. Further, a sandwich saturation protocol was employed in order to determine the affinity of an MCA for receptor-bound hCG. Rat testis membranes, preincubated with hCG, were exposed to increasing concentrations of ‘*“I-INN-bLH-1 as well as, in parallel, 1’SI-INN-hCG-42 (a sister-
Ka(MCAs): 0.1 33 nd 3 Ka(receptor): 286
1
1 15 0.1 2 0.3 11 1
1
3
Fig. 2. Epitope accessibility pattern of receptor-bound hCG (black bars) as probed by the receptor-hCG-““I-MC/\ sandwich approach using 14 different MCAs against 14 topographically distinct surface epitopes of hCG. As a positive control, i.e. for assessing the principal reactivity, the same preparation of each ‘z51-MCA was tested in parallel also on hCG bound to a capture MCA (stipped bars) of known compatibility (Schwarz et al., 1986, 1991; Berger et al., 1990); note that for most ‘2s1-MCAs responses of specific binding were greater than 15,000 cpm. The equilibrium affinity (K,, I.mol-’ X 10’) of each MCA for free hCG, determined at 4 ’ C by displacement with unlabeled hCG, is given underneath (rounded values taken from Berger et al., 1990; Schwarz et al.. 1991). For comparison, also the K,, of the rat testis hCG receptor for hCG is given (taken from Fig. 1). Background of iZf-MCA binding was assessed by omission of hCG, or by using equal amounts of rat liver instead of rat testis membranes. Each bar is the mean of duplicate determinations of a single experiment repeated twice.
O,i)2
3
50
100
lz51-MCA bound (fmole/mg protein)
a4 MCA.b~und hCG
0,oO 0
5 10 125 I-MCA bound (x 10 .‘* mole/L)
Fig. 3. Scatchard plot representations of two different MCAs specific for the /33 epitope of hCG, as obtained at 22°C in “‘I-MCA sandwich saturation experiments using either receptor-bound hCG (total incubation volume: 1 ml; left panel) or cu4 capture MCA-bound hCG (total incubation volume: 0.2 ml; right panel). Note that both /33-MCAs (i.e. INN-bLH-I, INN-hCG-42) recognize the same number of epitopes, i.e. 100 fmol/mg protein (=0.018 nM) (left panel). This B,,>,* concords with the number of hCG molecules bound, as determined in a saturation assay using ‘a51-hCG (not shown). Note further that the affinity of INN-bLH-1 is 9 times higher for recept~~r-bound hCG than for (~4 MCA-bound hCG. Each point in these plots is the mean of duplicate determinations of a single experiment repeated twice.
MCA also directed towards the /33 epitope: Berger et al., 1990). Scatchard plots shown in Fig. 3 (left panel) indicate that the affinity (I(,) of INN-bLH-1 is 91 k 8 x lo8 1. mol-‘, a number that is = 10 or = 90 times higher than the K,,of INN-bLH-1 for cy4-MCA-bound hCG (Fig. 3, right panel) or soluble hCG (i.e. = 1 X 10’ I * mol_ ‘, Fig. 2), respectively. INN-hCG-42, whose affinity for receptor-bound hCG was found to be = 16 times lower than that of INN-bLH-1 (K;$ = 5.6It0.2 X 10’ 1. mol-‘1 (Fig. 3, left panel), was still of sufficient strength as to recognize the same number of p3 epitopes on receptor-bound hCG as did INN-bLH-1. The J33 and /35 epitopes are adjacent on receptor-unbound hCG (Berger et al., 1990), because binding of the respective MCAs is mutually exclusive (see compatibility table shown elsewhere: Schwarz et al., 1991). To test whether this applies also for receptor-bound hCG, a self- and cross-inhibition experiment was performed. As shown in Fig. 4, ‘2’I-INN-bLH-1 (p3) binding to receptor-bound hCG could dose-dependently be
log [MCA], M Fig. 4. Inhibition of binding of the p3 epitope-specific “‘IMCA INN-bLI-I-I by increasing doses of unlabeled INN-bLH1 or INN-hCG-58, as determined in a sandwich self- and cross-inhibition experiment. Note that the MCA specific for the 83 epitop~-adjacent g5 epitope is incompatible with ‘2”I-MCA INN-bLl_S-1just as is INN-b~H-1 itself. Blank (zero point) was determined in hCG-preincubated membranes which were subsequent to washing exposed to ‘251-INN-bLH-l in the absence of INN-bLH-1 (maximal binding of “‘I-INNbLH-I). Each point is the mean of duplicate determinations of a single experiment repeated twice.
inhibited by unlabeled INN-hCG-58 (/35) and, as positive control, also by itself. Note that the reciprocal of the IC,, (1.5 x lo-” M) of INN-bLH1, i.e 67 X lo8 1. mol-‘, is comparable to its K, = 91 X 10s 1. mol-’ determined by saturation assay (Fig. 3, left panel). Discussion Based on our previous compatibility assessment using paired MCA binding (capture MCAhCG-‘25f-MCA sandwich), 14 epitopes were distinguished which are distributed in a characteristic topographical manner on the surface of hCG (Schwarz et al., 1986; Berger et al., 19901. Considering the known diameter of an antibody’s Fab cylinder on one hand (Novotny et al., 1983) and the Stokes radius of hCG on the other (Pierce and Parson, 1981), these 14 epitopes are very likely spread over a considerable portion of the water accessible surface of hCG rather than clustered in a small district (Schwarz et al., 1986). Hence, they can be utilized as coordinates or landmarks for testing hCG in its receptor-bound state. As shown in Fig. 2, 12 of 14 epitopes were invisible on receptor-bound hCG. Since incom-
plete receptor occupancy, insufficient MCA affinity and/or ‘reactivity’ could be ruled out by appropriate experiments, as discussed already above, only two factors remain to be considered as possible causes for the ‘loss’ of epitopes: (i) receptorinduced conformational change of hCG, and (ii) steric hindrance by the receptor. Despite hoio-hCG is structurally stabilized by a great number of disulfide bonds within its subunits (Pierce and Parsons, 1981), a change of hCG’s conformation within certain limits is still possible, such as following subunit association (Strickland and Puett, lYSZ), or deglycosylation, or, for that case, receptor binding. Consistent with obse~ations obtained by others indicating that such changes can be ‘sensed’ by antibodies (Keutmann et al., 1985; Norman et al., 1985; Hojo and Ryan, 1987; Moyle et al., 1987), we have reported on 2- to = 90-fold differences in MCA affinity for holo-hCG vs. the subunits (Berger et al., 19901, for native vs. deglycosylated hCG (Schwarz et al., 1991a1, and, as shown here (Fig. 31, for for vs. capture MCA-bound hCG or for free vs. receptor-bound hCG. We have also addressed the possibility that a conformational change could have led to ‘dislocation’ of epitopes, i.e. a change in the relative distances of two epitopes, resulting in change of the mutual compatibility of a pair of respective MCAs. Yet, alterations in the epitope topography could neither be detected between native and deglycosylated hCG (Schwarz et al., 1991a) nor between free and receptor-bound hCG (Fig. 4). Thus, ‘natural’ changes in the overall conformation can ‘reshape’ every or certain of hCG’s surface epitopes, leading to increase (e.g. INN-bLH-1, /33 epitope) but also decrease of MCA affinity (e.g. INN-hCG-58, p5 epitope), similar to what was observed by Moyle et al. (1987). However, unlike denaturation, such changes do not appear to be profound enough as to disrupt the antigenic topography or to destroy the antigenic integrity with consequent unreactiveness of MCAs. Thus, epitopes appear to be fairly robust landmarks of a globular protein’s surface. Otherwise, the intactness of the /33 and /35 epitopes would be extremely difficult to reconcile with the assumption that 12 of 14 epitopes could have been destroyed.
76
The experiment shown in Fig. 3 (left panel) indicates that INN-hCG-42, in spite of even 16fold lower affinity for receptor-bound hCG, could just as reliably detect the p3 epitope as its sister MCA INN-bLH-1. In addition, it is shown that every of the 14 different ‘2”1-MCAs could very we11 recognize hCG when bound to a compatible capture MCA (stippled bars in Fig. 21, whether being of high or low affinity (K, values ranging from 33 to 0.1 X 10’ 1 *moll’l. Thus, in any sequential sandwich protocol (be it receptor-hCG‘251-MCA or capture MCA-hCG-““I-MCA), the affinity of the detection MCA determines the degree of ““I-MCA binding but plays otherwise no decisive role, i.e. whether binding is possible at all or not. The reason is that in each of the incubations only two reaction partners are present, i.e. receptor and hCG in the first step and receptor-bound hCG and “‘1-MCA in the second. In addition, receptor-bound hCG does not dissociate and therefore not partition, so that also the relative affinity of the ““I-MCA is of little importance (INN-bLH-1 was reactive in spite of = 300 times lower K, compared to that of the receptor, see Fig. 2). Thus, the advantage of the sequential sandwich approach over protocols employing concurrent incubation of membranes with hCG and 12’1-MCA (or with ‘Z”I-hCG and MCA) is that it provides a simple all-or-none type end point, that is a response which is solely dependent on the compatibility between the two binding reagents (receptor and “‘I-MCA), i.e. freedom from steric hindrance exerted by the respective other reagent. This has previously clearly been shown in the capture MCA-hCG- “$1-MCA configuration where reciprocal combinations of two compatible MCAs yielded always identical results (Schwarz et al., 1986). Thus, the inaccessibility of 12 of 14 epitopes, as shown in Fig. 2, is most likely due to masking by moieties of the receptor. The LH/hCG receptor (McFarland et al., 1989) as well as the receptors for TSH (Parmentier et al., 1989) and FSH (Sprengel et al., 1990) have recently been cloned. Their EDNA-derived primary structures indicate that they share architecture and membrane topology with the G protein-coupled family of receptors (prototype being rhodopsin). However, unlike members that bind small ligands such as
adrenoceptors, the hCG receptor features in addition a long (341 residues) extracellular domain (consisting of 14 tandemly arranged leucine-rich repetitive motifs, presumably archetypic modules of protein-protein interaction). The seven transmembrane helices of all G protein-coupled receptors have been shown to be ring-like arranged (Frielle et al., 19881, thus f$rming a pocket whose inner diameter is = 15 A, thus Dable to fully accommodate a ligand of = 10 A length (e.g. epinephrinel (Strader et al., 1989) but, very probably, not a ligand of a Stokes radius of = 37 A (Pierce and Parsons, 1981) such as hCG. Hence, the only candidate moiety to bind hCG appears to be that long extracellular domain. Antibody labeling and site directed mutagenesis experiments have only recently verified that this domain is truly extracellular (Rodriguez and Segaloff, 1990) and that it binds hCG with an affinity comparable to the non-truncated hCG receptor (Xie et al., 1990; compare also Wadsworth et al., 1990 as to the TSH receptor). However, it is still not understood how and how much of the extracellular domain participates in forming the actual contact(s) with hCG: is it only a short stretch of the N-terminal portion or are muhiple stretches required that are themselves continuous or discontinuous? Peptide competition experiments have suggested the involvement of contact sequences spread over the entire length of this domain on one hand (especially residues 22-35) (Roche and Kitzmann, 1990) and of the hCG molecule on the other ((~1-15, n2646, ~~75-92, p38-57, /?93- 1001(Ryan et al., 19137). The question is, how is the extracehular domain folded to allow such a multifacetted interaction? The results of the here presented epitope accessibility paradigm may provide clues to such questions. The total area of all epitopes combined represents a certain fraction of and is thus representative for the total surface area of hCG. Hence, the fact that most (i.e. 12 of 14) epitopes were inaccessible suggests that moieties of the hCG receptor cover a considerabIe portion of the surface of hCG, relatively more of its cy than j3 subunit, as was suggested also by others (Ji and Ji, 1981; Moyle et al., 1982; Milius et al., 1983; Hwang and Menon, 1984). So much of hCG concealed seems difficult to reconcile with a com-
77
pactly folded superstructure of the extracellular domain (model A of Frazier et al., 1990). For the same but yet another reason also model B, implying immersion of the hormone into the transmembrane pocket, seems unlikely: given that the extracellular loops of the hCG receptor are not longer than those of the adrenoceptors, also the transmembrane pocket is likely to be of similar volume, that is, too small as to accommodate hCG.
Lp3,
We therefore propose a tentative model (schematically portrayed in Fig. 5) in which the extracellular domain is viewed as a rather open and flexible structure that reaches out into the extracellular space. As such it may first capture with its N-terminal part (e.g. residues 22-35 and some others) a certain part of hCG. In an adaptive, ‘hand-and-glove’-like mode of interaction, a sequence of multiple further discrete proteinprotein contacts is then established, as two obser-
ps epitopes accessible
Fig. 5. Schematic and tentative model of the differential modes of interaction of epinephrine (to the right) and of hCG (to the left) with their respective receptors representing prototypes of the neuronal and the endocrine class of G protein-coupled receptors, respectively. The putative structures of these receptors were taken from McFarland et al. (19891, Frielle et al. (1988) and Strader et al. (19891, and modified for incorporating the data obtained here by the epitope accessibility paradigm. hCG is depicted as a compact globular protein carrying I4 surface epitopes some of which being partly or fully on the back (i.e. tu3, cu5, @2, ~$41. The long extracellular domain of the hCG receptor is shown as an extended structure which in a ‘hand-and-glove’-like manner binds with high affinity and long occupancy hCG and thereby extensively covers the surface of hCG, i.e. masks most of its epitopes (for clarity, only accessible epitopes being shown; dark areas). This is in contrast to a ‘key-and-lock’-type, low affinity and short occupancy interaction of epinephrine and similar neurotransmitters with the transmembrane pocket of neuronal-type receptors. In both types, the third intracellular loop couples in an agonist-dependent manner with the a subunit of the G protein ((~/3y), thereby promoting (spy dissociation and subsequent activation of the adenylyl cyclase (AC) (Gilman, 1987). NANA, N-acetylneuraminic acid.
7x
vations suggest: (i) the relatively slow association kinetics (t,,2ass = 2 h), and (ii) the obvious perturbation sensitivity of this process: preincubation of 12”I-hCG with MCA abrogated the receptor-binding ability of ‘2SI-hCG-MCA complexes, no matter what part (epitope) of the surface of hCG was occupied (even the /33-MCA did so), as reported previously (Schwarz et al., 1988). At the end of this binding process, most of the epitopes of hCG are (passively) concealed by the ligand-binding moietv of the rece&or in which hCG has assumed a defined orientation. For, would the binding behavior be stochastic, many different orientations would be permitted, each with a different epitope accessibility pattern, so that, globally, all 14 epitopes would be accessible. Whether certain residues of either the hormone or the hormone-occupied extracellular domain (receptor autorecognition), interact in addition with the extracellular loops of the receptor, awaits further studies. This epitope accessibility paradigm has also been applied to the antagonist deglycosylated hCG in order to elucidate the role of the carbohydrate moieties on receptor interaction and signal transduction (Schwarz et al., 1991b). The observation that the p3 epitope was accessible on receptor-bound hCG may open important diagnostic and therapeutic possibilities, such as the use of this MCA as a means to (scintigraphically) visualize the degree of LH/ hCG receptor occupancy in vivo, or as a vehicle to specifically target a cytotoxic agent to a Leydig cell tumor expressing hCG receptors. Acknowledgements
This work was supported in part by the Austrian Academy of Sciences. We thank Ms. Regine Gerth, Renate Goerz, Irene Gaggl and Barbara Mayer for their skillful technical assistance and the National Pituitary Agency in Baltimore, MD, U.S.A., and Dr. W.E. Merz (University of Heidelberg, F.R.G.) for having kindIy supplied us with various glycoprotein hormone preparations. References Berger, P., Kofler, R. and Wick, Immunol. 5, 157-161.
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