Molecular and Cellular Endocrinology, 12 (1990) 63-70 Elsevier Scientific Publishers Ireland, Ltd.
MOLCEL
63
02317
Conformation of the a-subunit of glycoprotein hormones: a study using polyclonal and monoclonal antibodies Rajan R. Dighe, G. Satyanarayana Mm-thy, Basangowda and N. Raghuveer Moudgal
S. Kurkalli
Department of Biochemistry and Primate Research Laboratory, Indian Institute of Science, Bangalore 560012, India (Received
Key words: Glycoprotein
hormone;
Monoclonal
27 March
antibody;
1990; accepted
Polyclonal
antibody;
2 May 1990)
Conformation
of o-subunit
The conformation of the common a-subunit of human glycoprotein hormones, luteinizing hormone (hLH), follicle-stimulating hormone (hFSH), thyroid-stimulating hormone (hTSH) and chorionic gonadotropin (hCG) was probed using a highly specific polyclonal antiserum against the a-subunit of hCG and several monoclonal antibodies (MAbs) produced against hCG which recognized the a-subunit in free and combined form. The cu-subunit was found to be conformationally altered (compared to its conformation in the isolated state) when it was in combination with various P-subunits as indicated by shifts in the displacement curves of binding of [‘251]hCGa to the polyclonal antiserum. The extent of the change was dependent on the P-subunit present with minimum change being observed with hLHP, intermediate with hCGP and maximum change with hFSH and TSH P-subunits. However, the affinity constants of this antiserum for all four hormones were nearly similar. Further, it was also found that binding of any one of the glycoprotein hormones to this antibody could be completely inhibited by any other hormone suggesting that the conformation of the a-subunit in all the four hormones is probably very similar. This was further investigated using five hCG MAbs capable of recognizing the a-subunit, but with different epitope specificities. All these MAbs could recognize all the four hormones suggesting the presence of the epitopes in these proteins. These epitopes were conformation specific since the MAbs did not bind reduced and carboxymethylated cY-subunit. Displacement analysis using [ 125I]hCG as the tracer showed that two epitopes have nearly the same conformation in all the four hormones, while two were partially modified depending on the P-subunit present. Based on these results, it is concluded that the cY-subunit of glycoprotein hormones has nearly the same conformation, though subtle differences do exist.
Introduction The human glycoprotein hormones, hormone (hLH), follicle-stimulating
luteinizing hormone
Address for correspondence: Dr. R.R. Dighe, Department of Biochemistry and Primate Research Laboratory, Indian Institute of Science, Bangalore 650012, India. 0303-7207/90/$03.50
0 1990 Elsevier
Scientific
Publishers
Ireland,
(hFSH), thyroid-stimulating hormone (hTSH) and chorionic gonadotropin (hCG) have an identical a-subunit noncovalently linked to hormonespecific /?-subunit (Pierce and Parsons, 1981; Sairam, 1983). Specific antibodies against specific regions of these hormones have revealed several interesting features of the proteins as well as the nature of interactions between the hormone and Ltd.
64
the receptor (Dighe and Moudgal, 1981, 1983; Moyle et al., 1982; Milius et al., 1983). Earlier studies from our laboratory using highly specific antibodies against the 01- and P-subunits of hCG have clearly shown that the initial binding of hCG to the receptor takes place mainly through the P-subunit of the hormone (Dighe and Moudgal, 1981, 1983). A similar mechanism appears to be true for FSH also (Moudgal et al., 1989). However, the role of the a-subunit in binding of the hormone to the receptor could not be ascertained, but other studies have shown that the integrity of the a-subunit appears to be crucial for expression of biological activity of the glycoprotein hormones (Sairam, 1983). Hence it was of interest to find out if the conformation of the a-subunit in the four human glycoprotein hormones was similar, if not identical. In the present study an attempt has been made to investigate the conformation of the (Ysubunit in these hormones using polyclonal and monoclonal antibodies against the a-subunit of hCG.
iodination reaction was initiated by addition of 50 pg of chloramine T in a total reaction volume of 65 ~1, allowed to proceed for 45-60 s on ice and terminated by addition of 800 pg of ,0mercaptoethylamine. Iodogen iodination reaction was carried out on ice for 1.5 min using 25 pg of iodogen. The unincorporated radioactivity was separated from the incorporated 1251 by gel filtration on Sephadex G75. The specific activity of each preparation was determined by trichloroacetic acid (TCA) precipitation and ranged between 30,000 and 50,000 cpm/ng of protein. Reduction and carboxymethylation of hCGa hCGa (2 mg/ml) was dissolved in 6 M guanidine hydrochloride and reduced with 50-fold excess of dithiothreitol overnight at room temperature. Carboxymethylation was carried out by addition of excess of iodoacetic acid. Reduced and carboxymethylated hCGa (RCM hCGa) was recovered by dialysis against distilled water and lyophilization. Radioiodination of RCM hCGa was carried out by the chloramine T method.
Materials and methods Hormones and chemicals hCG (CR123) and its LY-and p- (CR123) subunits were obtained from Dr. Canfield through the National Hormone Pituitary Program and the Center for Population Research of the National Institute of Child Health and Human Development, NIH, U.S.A. hLH, hFSH and hTSH radioimmunoassay (RIA) kits were obtained from NIAMDD through the Institute of Research in Reproduction, Bombay. The iodination grade hLH and hTSH from these kits were also used as displacing agents. hFSH was obtained from Dr. A.F. Parlow (AFP 1589A). [‘251]NaI was purchased from the Amersham Radiochemical Company, U.K. All other chemicals were of analytical grade.
Polyclonal antiserum Polyclonal antiserum was raised against the a-subunit of hCG (hCGa a/s) in the rabbit as described earlier (Dighe and Moudgal, 1983). Aliquots (100 ~1) of various dilution of this serum were incubated with ‘251-labelled hCG, hCGa and hCGP (approximately 100,000 cpm of each labelled probe) overnight at room temperature (27-28” C). The antigen-antibody complex was precipitated by addition of goat antirabbit IgG and polyethylene glycol 6000 (2.5%) centrifuged at 5000 x g for 20 min and the pellet counted for radioactivity. The nonspecific binding was determined by incubating the labelled probes with normal rabbit serum and determining the radioactivity in the pellet.
Iodination of hormones All the hormones and the subunits used in this study were iodinated either using the chloramine T method according to the procedure of Hunter and Greenwood (1962) or using Iodogen (Fraker and Speck, 1978). Usually 5 pg of each protein was iodinated with approximately 0.5 mCi of [‘251]NaI. In case of the chloramine T method, the
Monoclonal antibodies Several MAbs against hCG were produced and characterized as described elsewhere (Dighe et al., 1990). Briefly, splenocytes obtained from BALB/c mice immunized with hCG were fused with SP2/0 cells using polyethylene glycol 3000 as the fusogen. Hybrids were selected in medium containing hypoxanthine, aminopterin, and thymidine. One
65
week after the fusion the culture supernatants were tested for presence of hCG antibodies by incubating 100-200 ~1 culture supernatant with approximately 100,000 cpm of [‘251]hCG overnight at room temperature. The nonspecific binding was determined by incubating labelled hCG with SP2/0 spent medium. The antigen-antibody complex was precipitated by goat antimouse IgG and polyethylene glycol 6000 (2.5%) centrifuged and the radioactivity in the pellet was counted in an LKB autogamma counter. Those hybrids which showed hCG binding were expanded first into 24-well plates and then into bottles. The subunit specificity of each clone was established by inof hCG cubating ‘251-labelled (Y- and /&subunits with the culture supernatants. The clones which showed binding to hCG and hCG a-subunit were chosen for this study. The monoclonality of these clones, as far as binding to hCG was concerned, was established as described elsewhere (Dighe et al., 1990) and some of the clones were subcloned by limiting dilution.
lo-
E 8
m-c
hCGa
H
KG
0-c
hLH
x-x
hSSH
A--a
hFSH
01
10
10
lix3
Fig. 1. Displacement of binding of [‘251]hCGa to hCGcv a/s by hCGa and human glycoprotein hormones. [‘251]hCG LYsubunit (approximately 150,000 cpm) was incubated with hCGa a/s (final dilution) in a total volume of 0.5 ml in the presence of 0.5-100 ng of each displacing agent overnight at room temperature. The antigen-antibody complex was precipitated by polyethylene glycol-double antibody method and radioactivity in the pellet was counted in an LKB autogamma counter. Each value is the mean of triplicates.
Results Aliquots (100 ~1) of various dilutions of the hCGa a/s were incubated with [‘251]hCG, [‘251]hCG a and [ 1251]hCGfi and binding was determined as described in Materials and Methods. It was found that this a/s could bind both hCG and hCGa at a very high dilution (l/500,000), but was able to bind [‘251]hCG/3 only at the lowest dilutions (l/500 and l/2500). This showed that the (x a/s was highly specific for the a-subunit and at the dilutions used in the later experiments (l/250,000), P-subunit of hCG did not bind or interfere at all. Changes in the conformation of the antigenic determinants in the a-subunit following recombination with various /&subunits were next quantified by establishing displacement curves for all the antigens with [1251]hCGa as the tracer. The hCGa a/s (l/250,000 dilution) was incubated with [‘251]hCGa in the presence of hCGcw, hCG, hFSH, hTSH and hLH overnight at room temperature and binding was determined as described above. The EC,, of each displacement curve was obtained by using a four-parameter logistic program written for the Apple IIe computer. As shown in Fig. 1
and Table 1, the a-subunit undergoes marked changes in the conformation when it is in combination with various P-subunits as shown by shifts in the EC,,. Thus, while the EC,, for hCGa was 108 k 4 fmol, the EC,, for the intact hormones was always higher. The cu-subunit in hLH appeared to have undergone the least changes from its free state conformation as the EC,, for the hLH in displacement analysis was 166 + 6 fmol
TABLE
1
ANALYSIS OF DISPLACEMENT OF [“‘I]hCGa BINDING TO hCGa a/s BY GLYCOPROTEIN HORMONES: CONCENTRATION REQUIRED TO OBTAIN 50% DISPLACEMENT EC,, was obtained by analyzing Fig. 1 using the four-parameter the Apple IIe computer. Displacing hCGa hCG hLH hFSH hTSH
agent
the binding data presented in logistic equation written for
EC,,
(fmol f SE)
108k 4 278* 6 166* 6 649k18 735 f 39
66 TABLE
2
ASSOCIATION AND hCGci HORMONES
CONSTANTS OF THE hCGa a/s FOR ALL THE HUMAN GLYCOPROTEIN
hCGa a/s (1/250,000) was incubated with labelled antigens in the presence of varying concentrations of the respective hormone/subunit overnight at room temperature and the binding was determined. The binding data were later converted into Scatchard plots. Hormone/subunit
K, (Mm’)
hCGa hCG hLH
4.3 3.18 1.2 6.39 3.4
hFSH hTSH
XlO’O x 109 xlo’c x lo9 x109
while that for hCG was 278 f 6 fmol. In contrast, the same subunit appears to have undergone relatively more changes when the fi-subunit was either hFSH or hTSH, the EC,, in both cases being approximately 700 fmol. Determination of affinity constants of hCGa a/s for each glycoprotein hormone The hCGa a/s was incubated with each iodinated probe in the presence and absence of the respective unlabelled antigen and the binding data were converted into Scatchard plots (Scatchard, 1949). As shown in Table 2, affinity constants of this a/s for all the four glycoprotein hormones are nearly the same although the affinity for hLH was relatively higher compared to the other hormones. The affinity for hCGcY was much higher compared to all the glycoprotein hormones. Displacement of ‘2sI-labelled hormones by other hormones The hCGa a/s was incubated with ‘251-labelled hCG/hLH/hFSH in the presence of unlabelled hCG, hLH, hFSH and hCGa and binding of the labelled antigen to the a/s at a dilution of l/50,000 was determined. As shown in Fig. 2, each of these iodinated probes could be completely displaced by all other antigens suggesting that the antigenic determinants are conserved in all the three glycoprotein hormones used in this experiment.
Binding of MAbs to iodinated probes MAbs against hCG were produced and characterized as described elsewhere (Dighe et al., 1990). Five MAbs specific for the cx-subunit, but having different epitope specificities, were selected for these studies. All the five MAbs could bind [‘251]hCG, hLH, hFSH and hTSH, as well as hCG a-subunit suggesting the presence of the epitope in these proteins (Dighe et al., 1990). To determine if these MAbs were conformation specific, hCGa was reduced and carboxymethylated and then radioiodinated (RCM hCGa). None of these MAbs could bind to the RCM hCGa suggesting the conformation-specific nature of these MAbs. In order to determine to what extent the epitope recognized by MAbs in hCG are conserved in other glycoprotein hormones, displacement analysis was carried out. MAbs were adsorbed on to a plastic tube through an immunochemical bridge as described elsewhere (Murthy et al., 1989). Briefly, plastic tubes were coated with normal mouse serum which was then saturated with antimouse IgG raised in the goat. The tubes were finally coated with MAb at appropriate concentrations. Each MAb (solid phased) was incubated with [‘*‘I]hCG in the presence of all the four hormones, the a-subunit as well as RCM hCG a-subunit and binding was determined after overnight incubation at room temperature. The binding data were analyzed with the four-parameter logistic equation program and the EC,o determined by the computer are shown in Table 3. It was found that RCM hCG a-subunit was unable to displace hCG from binding to any of the MAbs again confirming that these MAbs are conformation specific and do not recognize any sequence in the a-subunit. It was also clear that the displacement of the labelled hCG varied depending on the MAb and displacing agent. Thus, the epitope recognized by the MAb 52/8 was present in all the glycoprotein hormones and appears to be present in nearly the same conformation as demonstrated by identical EC,, for all the antigens except hTSH which showed a higher ECSo. The other epitope that appeared to be present in a nearly similar conformation was the epitope recognized by the MAb 52/21. The epitope recognized by MAb 52/2 seems to be more specific for hCG as shown by its relatively higher affinity for hCG. Similarly affin-
67
I-
3-
3-
61O-
O-
125
10 O-
I
hLH
I3o-
6 Os x
4O- -
cl0 \ m _ 2 0.
M
hCG
U-0
hLH
A-A
hFSH
O-O
hCG
\
ng disploclng
Fig. 2. Displacement of binding incubated with [ 125 I] hCG (Fig. overnight at room temperature determined by incubating each concentration of the unlabelled
\
alpha
agent
of [‘251]hCG/hLH/hFSH to hCGn a/s by hCGa, hCG, hLH and hFSH. hCGa a/s (l/50,000) was 2a), [‘251]hLH (Fig. 2b) and [ ‘251]hFSH (Fig. 2c) in the presence of hCG, hLH hFSH and hCGa and binding was determined as described in Materials and Methods. The nonspecific binding was labelled antigen with normal rabbit serum and was subtracted from the binding observed with each hormone. The binding observed in the absence of any unlabelled antigen was termed & and considered as 100%. The values are means of duplicates.
68
TABLE
3
ANALYSIS OF DISPLACEMENT OF BINDING OF [‘251]hCG TO MAbs BY GLYCOPROTEIN CENTRATION REQUIRED TO OBTAIN 50% DISPLACEMENT (PMOL* SE/TUBE)
HORMONES:
CON-
MAbs were adsorbed on to a plastic tube through an immunochemical bridge. Each MAb was incubated with [‘251]hCG in the presence of 10-1000 ng of each displacing agent and binding was determined after overnight incubation at room temperature. The binding data were analyzed on an Apple IIe computer using the four-parameter logistic equation. The numbers in parentheses indicate the 95% confidence limits of the EC,,. MAb
hCG
hCGa
hLH
hFSH
hTSH
52/2
2.44*0.2 (2.0-2.9) 1.55 f 0.1 (1.4-1.72) No dis.
No dis. a
No dis.
No dis.
No dis.
1.48+0.1 (1.32-1.72) 0.9*0.1 (0.7-1.14) 8.45 f 0.8 (7-10.3) 10.96 + 0.25 (10.4-11.48)
2.2*0.16 (1.9-2.56) 13.7kO.8 (12.2-15.4) 2.4 + 0.2 (2.07-2.83) 5.5 +0.14 (5.2-5.8)
1.5*0.1 (1.38-1.76) 14.7*1.2 (12.3-17.5) 5.1 * 0.4 (4.38-6) 30.5 + 1.83 (26.9-34.5)
8.14kO.43 (8.14-9.08) No dis.
52/8 52/11 52/21 G,,,/f,
3.9 + 0.27 (3.34-4.46) 0.76 f 0.04 (0.69-0.94)
a No dis. = less than 50% displacement hCGa.
obtained
with 1 ng_ of the displacing
ity of MAb G,,/f, was significantly higher for hCG than that for hLH, hFSH and hCG&. The affinity of MAb 52/11 was surprisingly higher for the isolated subunit than for hLH and hFSH which in turn was much higher than that for hCG. Discussion It is known that the subunits of glycoprotein hormones undergo considerable conformational change when they combine to form the native, biologically active hormone (Ajoj et al., 1973; Ingham et al., 1973; Bewley et al., 1974; Bewley, 1978). However, these changes were of a gross nature as shown by changes in the circular dichroism spectrum or increase in the binding of anilino-naphtho-sulfonic acid to the hormone and did not yield any information about changes in the specific regions of the hormone molecule. In this study we investigated changes in the a-subunit of glycoprotein hormones when it is in combination with various P-subunits of the glycoprotein hormones using polyclonal and monoclonal antibodies as the probes. The polyclonal antiserum used in this study is highly specific for the a-subunit and had no cross-reactivity with hCG P-subunit. The cu-subunit was found to undergo conformational change
agent.
No displacement
35.6 +4 (28.16-45) No dis.
was obserued with
I pg RCM
when in combination with the P-subunit. This was shown by demonstrating shifts in the displacement curve. The shift depended on the P-subunit with which the a-subunit was present. Thus, while with hLH/I the a-subunit seemed to have undergone the least change, it has undergone relatively more changes when the P-subunit was either of FSH or TSH. However, the fact that this a/s binds to the a-subunit in all the four a-/3 dimers with nearly the same affinity suggests that the conformation of the a-subunit in all the hormones must be nearly similar. The proof for this possibility is provided by the observation that it was possible to completely displace binding of [ 1251]hCG/ hLH/ hFSH by either of these agents even at lower dilutions of the a/s. These results again imply that conformation of the a-subunit in the four human hormones must be very similar. However, the above results point out only gross similarities in the conformation of the a-subunit in the glycoprotein hormones. Hence these were followed by studies with MAbs. The studies carried out with the MAbs have revealed several interesting features. The MAbs used in this study were raised against hCG, but were specific for the a-subunit of hCG. These MAbs recognized unique conformation of the antigen and did not recognize any specific sequences as indicated by absence of
69
binding of RCMa to any of the MAbs. All these MAbs recognized and could bind ‘251-labelled human glycoprotein hormones and the a-subunit. This showed the existence of the common epitopes in all the four glycoprotein hormones. Displacement analysis with all the five MAbs using [‘251]hCG as the tracer showed that there are distinct similarities in many of the epitopes. The epitope recognized by MAb 52/8 appears to be identical in all the four giycoprotein hormones since hCG, hFSH, hLH and hCGa: were able to displace [‘25]hCG with equal efficacy. Thus this epitope is expressed in the a-subunit but is not altered as a result of recombination with any of the P-subunits. The epitope 52/21 was also conserved in all the antigens, although it was modified as indicated by significant shifts in the EC,,. The epitope recognized by Mab G,,/f, is similarly conserved in hCG, hLH, hFSH and hCG&, but its affinity for hCG was much higher than that for other hormones. The affinity of MAb 52/2 for hCG appeared to be very high for hCG relative to other hormones as no displacement with any other hormone could be observed suggesting that this epitope is altered in other a-@ dimers. However, it must be pointed out that all the four glycoprotein hormones and the a-subunit bind to all the MAbs (Dighe et al., 1990) suggesting that the epitope is present in all the four hormones although with slightly altered conformation. The most surprising results were obtained with MAbs 52/11. This MAb seems to have very high affinity for hCGa, hLH and hFSH than that for hCG. The reasons for this result are not presently known, but a different MAb recognizing the epitope very close to that recognized by this MAb also shows similar displacement curves (data not shown). The above results show that there are gross similarities in the conformation of the a-subunits in the four glycoprotein hormones. This is demonstrated by the use of polyclonal antibodies. Similarly many epitopes are conserved in all the four glycoprotein hormones. This is particularly true of the epitopes recognized by MAbs 52/8 and 52/21. However, there are dissimilarities also as indicated by shifts in the displacement curves of other MAbs. The dissimilarities in epitopes have been reported by Hojo and Ryan (1985) with two monoclonal
antibodies raised against hFSH but also capable of binding other glycoprotein hormones. However, both these MAbs appear to be recognizing the same epitope as their characteristics are nearly the same. In contrast, we have five MAbs with different characteristics as shown by different displacement curves, as well as binding characteristics (Dighe et al., 1990). Similar conformations of the a-subunit suggest several interesting possibilities. The four glycoprotein hormones have an identical a-subunit. There are a large number of reports indicating that the integrity of the a-subunit is absolutely essential for expression of biological activity of the hormones (see Sairam, 1983). The similar nature of the epitopes in the a-subunit in all glycoprotein hormones further underscores the importance of the a-subunit. It is tempting to speculate that the conserved conformation of the epitopes in the a-subunit of the native hormones plays an important role in the hormone action, viz. stimulation of G proteins and activation of adenylyl cyclase in the respective target organs, which is a common mechanism of hormone action. It can be hypothesized that the primary role of the hormone-specific P-subunit is to recognize the receptor and bring the a-subunit in proper orientation so that it can interact with regions of the receptor which are involved in signal transduction. In other words, the information necessary to stimulate the G proteins resides in the a-subunit. Our earlier studies with hCG and ovine FSH have clearly shown that the initial interaction between the hormones and their receptors takes place initially through the ,&subunits (Dighe and Moudgal, 1983; Moudgal et al., 1989). Although the inability of the a-specific MAbs as well as polyclonal antibodies to bind to receptor-bound hormone has been reported (Moyle et al., 1982; Milius et al., 1983), these experiments were carried out with a previously formed hormone-receptor complex and the time course of binding was never investigated as carried out by us (Dighe and Moudgal, 1983). Thus, the role of the P-subunit is probably to deliver the a-subunit, which by itself cannot bind to the receptor (Dighe et al., 1979), to the receptor site which, in turn, stimulates the response in the target organ. The ability of synthetic peptides from the a-subunit to stimulate the steroidogenic response is in total agreement with this concept
70
(Ryan et al., 1987). The recent demonstration of the importance of the glycosylation site at asparagine 52 (Matzuk et al., 1989) and involvement of histidine residues (Willey and Leidenberger, 1989) of the a-subunit in stimulation of response further underscores the importance of the a-subunit in hormone action. The precise localization of the epitopes in the a-subunit would certainly help in understanding the signal transduction mechanism. Several attempts have been made in the literature to locate these epitopes using synthetic peptides (Bidart et al., 1987, 1988, 1989; Troalen et al., 1988). We have already located some of these epitopes in the hCG molecule (Murthy et al., manuscripts in preparation). We also have observed that the MAbs against hCG &subunit are relatively more effective blockers of hormone-receptor interaction than the aspecific MAbs (Dighe, unpublished observations) which again indicates the role of P-subunit in binding of the hormone to the receptor. Another interesting point that can be highlighted from these studies is that although the P-subunits of hCG and hLH are very similar and both bind to the same receptor, there are subtle differences in the folding of the a-subunit in these dimers. This may perhaps explain the relatively higher potency as well as affinity of hCG for its receptors compared to other LHs (Sheela Rani and Moudgal, 1985). Acknowledgements
The work reported here was supported by grants provided by the Department of Biotechnology, Government of India and the Indian Council of Medical Research, New Dehli. References Ajoj, S.M., Edlehoch, H., Ingham, K.C., Morgan, F.C.. Canfield, R.E. and Ross, G.T. (1973) Arch. Biochem. Biophys. 159,497-504.
Bewley, T.A. (1979) Recent Prog. Horm. Res. 35, 135-213. Bewley, T.A., Sairam, M.R. and Li. C.H. (1974) Arch. Biothem. Biophys. 163, 625. Bidart, J.M., Troalen, F., Bohuon, C., Hennen, G. and Bellet, D.H. (1987) J. Biol. Chem. 262,15483-15489. Bidart, J.M., Troalen, F., Bousfield, G.R., Biekrn, S. and Bellet, D.H. (1988) J. Biol. Chem. 263, 10364-10369. Bidart, J.M., Troalen, F., Bousfield, G.R., Bohuon, C. and Bellet, D.H. (1989) Endocrinology 124, Y23-989. Diphe, R.R. and Moudgal, N.R. (1981) in Functional Correlates of Hormone Receptors in Reproduction (Mahesh, V.B., Maldoon, T.G., Saxena, B.B. and Sadler, W.A., eds.), pp. 545-549, Eisevier/No~h-Holland, Amsterdam. Digbe, R.R. and Moudgal, N.R. (1983) Arch. Biochem. Biophys. 225, 490-499. Dighe, R.R., Muralidhar, K. and Moudgal, N.R. (1979) Biothem. J. 180, 573-580. Dighe, R.R., Murthy, G.S. and Moudgal, N.R. (1990) J. Immunol. Methods (in press). Hojo, H. and Ryan, R.J. (1985) Endocrinology 117,2428-2434. Ingham, K.C., Ajoj. SM. and Edelhoch, M. (1973) Arch. Biochem. Biophys. 159, 596-605. Matzuk, M.M., Keene, J.L. and Boime, I. (1989) J. Biol. Chem. 264, 2409-2414. Milius, R.P., Midgeley, A.R. and Birken, S. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 7375-7378. Moudgal, N.R., Sairam. M.R. and Dighe, R.R. (1989) J. Biosci. 14,91-100. Moyle, W.R., Ehrlich, P.H. and Canfield, R.E. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2245-2249. Murthy, G.S., Laxmi, B.S. and Moudgal, N.R. (1989) J. Biosci. 14, 9-20. Pierce, J.G. and Parsons, T.F. (1981) Annu. Rev. Biochem. 50, 465. Ryan, R.J., Keutmann, H.T., Charlsworth. M.C.. McCormick, D.J., Milius, R.P., Calvo, F.O. and Vutyavanich, T. (1987) Recent Prog. Horm. Res. 47, 383-429. Sairam, M.R. (1983) in Hormones and Hormonal Peptides. Vol. 11 (Li, Ch., ed.), pp. l-79. Academic Press, New York. Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 51,660-662. Sheela Ram, C.S. and Moudgal, N.R. (1985) Endocrinology, 116, 597-603. Troalen, F., Bellet, D.H., Ghillani, P., Puisieux, A., Bohuon, C.J. and Bidart, J.M. (1988) J. Biol. Chem. 263, 10370-10376. Willey, K.P. and Leidenberger, F. (1989) J. Biol. Chem. 264. 19716-19729.
MOLCEL
63
02317
Conformation of the a-subunit of glycoprotein hormones: a study using polyclonal and monoclonal antibodies Rajan R. Dighe, G. Satyanarayana Mm-thy, Basangowda and N. Raghuveer Moudgal
S. Kurkalli
Department of Biochemistry and Primate Research Laboratory, Indian Institute of Science, Bangalore 560012, India (Received
Key words: Glycoprotein
hormone;
Monoclonal
27 March
antibody;
1990; accepted
Polyclonal
antibody;
2 May 1990)
Conformation
of o-subunit
The conformation of the common a-subunit of human glycoprotein hormones, luteinizing hormone (hLH), follicle-stimulating hormone (hFSH), thyroid-stimulating hormone (hTSH) and chorionic gonadotropin (hCG) was probed using a highly specific polyclonal antiserum against the a-subunit of hCG and several monoclonal antibodies (MAbs) produced against hCG which recognized the a-subunit in free and combined form. The cu-subunit was found to be conformationally altered (compared to its conformation in the isolated state) when it was in combination with various P-subunits as indicated by shifts in the displacement curves of binding of [‘251]hCGa to the polyclonal antiserum. The extent of the change was dependent on the P-subunit present with minimum change being observed with hLHP, intermediate with hCGP and maximum change with hFSH and TSH P-subunits. However, the affinity constants of this antiserum for all four hormones were nearly similar. Further, it was also found that binding of any one of the glycoprotein hormones to this antibody could be completely inhibited by any other hormone suggesting that the conformation of the a-subunit in all the four hormones is probably very similar. This was further investigated using five hCG MAbs capable of recognizing the a-subunit, but with different epitope specificities. All these MAbs could recognize all the four hormones suggesting the presence of the epitopes in these proteins. These epitopes were conformation specific since the MAbs did not bind reduced and carboxymethylated cY-subunit. Displacement analysis using [ 125I]hCG as the tracer showed that two epitopes have nearly the same conformation in all the four hormones, while two were partially modified depending on the P-subunit present. Based on these results, it is concluded that the cY-subunit of glycoprotein hormones has nearly the same conformation, though subtle differences do exist.
Introduction The human glycoprotein hormones, hormone (hLH), follicle-stimulating
luteinizing hormone
Address for correspondence: Dr. R.R. Dighe, Department of Biochemistry and Primate Research Laboratory, Indian Institute of Science, Bangalore 650012, India. 0303-7207/90/$03.50
0 1990 Elsevier
Scientific
Publishers
Ireland,
(hFSH), thyroid-stimulating hormone (hTSH) and chorionic gonadotropin (hCG) have an identical a-subunit noncovalently linked to hormonespecific /?-subunit (Pierce and Parsons, 1981; Sairam, 1983). Specific antibodies against specific regions of these hormones have revealed several interesting features of the proteins as well as the nature of interactions between the hormone and Ltd.
64
the receptor (Dighe and Moudgal, 1981, 1983; Moyle et al., 1982; Milius et al., 1983). Earlier studies from our laboratory using highly specific antibodies against the 01- and P-subunits of hCG have clearly shown that the initial binding of hCG to the receptor takes place mainly through the P-subunit of the hormone (Dighe and Moudgal, 1981, 1983). A similar mechanism appears to be true for FSH also (Moudgal et al., 1989). However, the role of the a-subunit in binding of the hormone to the receptor could not be ascertained, but other studies have shown that the integrity of the a-subunit appears to be crucial for expression of biological activity of the glycoprotein hormones (Sairam, 1983). Hence it was of interest to find out if the conformation of the a-subunit in the four human glycoprotein hormones was similar, if not identical. In the present study an attempt has been made to investigate the conformation of the (Ysubunit in these hormones using polyclonal and monoclonal antibodies against the a-subunit of hCG.
iodination reaction was initiated by addition of 50 pg of chloramine T in a total reaction volume of 65 ~1, allowed to proceed for 45-60 s on ice and terminated by addition of 800 pg of ,0mercaptoethylamine. Iodogen iodination reaction was carried out on ice for 1.5 min using 25 pg of iodogen. The unincorporated radioactivity was separated from the incorporated 1251 by gel filtration on Sephadex G75. The specific activity of each preparation was determined by trichloroacetic acid (TCA) precipitation and ranged between 30,000 and 50,000 cpm/ng of protein. Reduction and carboxymethylation of hCGa hCGa (2 mg/ml) was dissolved in 6 M guanidine hydrochloride and reduced with 50-fold excess of dithiothreitol overnight at room temperature. Carboxymethylation was carried out by addition of excess of iodoacetic acid. Reduced and carboxymethylated hCGa (RCM hCGa) was recovered by dialysis against distilled water and lyophilization. Radioiodination of RCM hCGa was carried out by the chloramine T method.
Materials and methods Hormones and chemicals hCG (CR123) and its LY-and p- (CR123) subunits were obtained from Dr. Canfield through the National Hormone Pituitary Program and the Center for Population Research of the National Institute of Child Health and Human Development, NIH, U.S.A. hLH, hFSH and hTSH radioimmunoassay (RIA) kits were obtained from NIAMDD through the Institute of Research in Reproduction, Bombay. The iodination grade hLH and hTSH from these kits were also used as displacing agents. hFSH was obtained from Dr. A.F. Parlow (AFP 1589A). [‘251]NaI was purchased from the Amersham Radiochemical Company, U.K. All other chemicals were of analytical grade.
Polyclonal antiserum Polyclonal antiserum was raised against the a-subunit of hCG (hCGa a/s) in the rabbit as described earlier (Dighe and Moudgal, 1983). Aliquots (100 ~1) of various dilution of this serum were incubated with ‘251-labelled hCG, hCGa and hCGP (approximately 100,000 cpm of each labelled probe) overnight at room temperature (27-28” C). The antigen-antibody complex was precipitated by addition of goat antirabbit IgG and polyethylene glycol 6000 (2.5%) centrifuged at 5000 x g for 20 min and the pellet counted for radioactivity. The nonspecific binding was determined by incubating the labelled probes with normal rabbit serum and determining the radioactivity in the pellet.
Iodination of hormones All the hormones and the subunits used in this study were iodinated either using the chloramine T method according to the procedure of Hunter and Greenwood (1962) or using Iodogen (Fraker and Speck, 1978). Usually 5 pg of each protein was iodinated with approximately 0.5 mCi of [‘251]NaI. In case of the chloramine T method, the
Monoclonal antibodies Several MAbs against hCG were produced and characterized as described elsewhere (Dighe et al., 1990). Briefly, splenocytes obtained from BALB/c mice immunized with hCG were fused with SP2/0 cells using polyethylene glycol 3000 as the fusogen. Hybrids were selected in medium containing hypoxanthine, aminopterin, and thymidine. One
65
week after the fusion the culture supernatants were tested for presence of hCG antibodies by incubating 100-200 ~1 culture supernatant with approximately 100,000 cpm of [‘251]hCG overnight at room temperature. The nonspecific binding was determined by incubating labelled hCG with SP2/0 spent medium. The antigen-antibody complex was precipitated by goat antimouse IgG and polyethylene glycol 6000 (2.5%) centrifuged and the radioactivity in the pellet was counted in an LKB autogamma counter. Those hybrids which showed hCG binding were expanded first into 24-well plates and then into bottles. The subunit specificity of each clone was established by inof hCG cubating ‘251-labelled (Y- and /&subunits with the culture supernatants. The clones which showed binding to hCG and hCG a-subunit were chosen for this study. The monoclonality of these clones, as far as binding to hCG was concerned, was established as described elsewhere (Dighe et al., 1990) and some of the clones were subcloned by limiting dilution.
lo-
E 8
m-c
hCGa
H
KG
0-c
hLH
x-x
hSSH
A--a
hFSH
01
10
10
lix3
Fig. 1. Displacement of binding of [‘251]hCGa to hCGcv a/s by hCGa and human glycoprotein hormones. [‘251]hCG LYsubunit (approximately 150,000 cpm) was incubated with hCGa a/s (final dilution) in a total volume of 0.5 ml in the presence of 0.5-100 ng of each displacing agent overnight at room temperature. The antigen-antibody complex was precipitated by polyethylene glycol-double antibody method and radioactivity in the pellet was counted in an LKB autogamma counter. Each value is the mean of triplicates.
Results Aliquots (100 ~1) of various dilutions of the hCGa a/s were incubated with [‘251]hCG, [‘251]hCG a and [ 1251]hCGfi and binding was determined as described in Materials and Methods. It was found that this a/s could bind both hCG and hCGa at a very high dilution (l/500,000), but was able to bind [‘251]hCG/3 only at the lowest dilutions (l/500 and l/2500). This showed that the (x a/s was highly specific for the a-subunit and at the dilutions used in the later experiments (l/250,000), P-subunit of hCG did not bind or interfere at all. Changes in the conformation of the antigenic determinants in the a-subunit following recombination with various /&subunits were next quantified by establishing displacement curves for all the antigens with [1251]hCGa as the tracer. The hCGa a/s (l/250,000 dilution) was incubated with [‘251]hCGa in the presence of hCGcw, hCG, hFSH, hTSH and hLH overnight at room temperature and binding was determined as described above. The EC,, of each displacement curve was obtained by using a four-parameter logistic program written for the Apple IIe computer. As shown in Fig. 1
and Table 1, the a-subunit undergoes marked changes in the conformation when it is in combination with various P-subunits as shown by shifts in the EC,,. Thus, while the EC,, for hCGa was 108 k 4 fmol, the EC,, for the intact hormones was always higher. The cu-subunit in hLH appeared to have undergone the least changes from its free state conformation as the EC,, for the hLH in displacement analysis was 166 + 6 fmol
TABLE
1
ANALYSIS OF DISPLACEMENT OF [“‘I]hCGa BINDING TO hCGa a/s BY GLYCOPROTEIN HORMONES: CONCENTRATION REQUIRED TO OBTAIN 50% DISPLACEMENT EC,, was obtained by analyzing Fig. 1 using the four-parameter the Apple IIe computer. Displacing hCGa hCG hLH hFSH hTSH
agent
the binding data presented in logistic equation written for
EC,,
(fmol f SE)
108k 4 278* 6 166* 6 649k18 735 f 39
66 TABLE
2
ASSOCIATION AND hCGci HORMONES
CONSTANTS OF THE hCGa a/s FOR ALL THE HUMAN GLYCOPROTEIN
hCGa a/s (1/250,000) was incubated with labelled antigens in the presence of varying concentrations of the respective hormone/subunit overnight at room temperature and the binding was determined. The binding data were later converted into Scatchard plots. Hormone/subunit
K, (Mm’)
hCGa hCG hLH
4.3 3.18 1.2 6.39 3.4
hFSH hTSH
XlO’O x 109 xlo’c x lo9 x109
while that for hCG was 278 f 6 fmol. In contrast, the same subunit appears to have undergone relatively more changes when the fi-subunit was either hFSH or hTSH, the EC,, in both cases being approximately 700 fmol. Determination of affinity constants of hCGa a/s for each glycoprotein hormone The hCGa a/s was incubated with each iodinated probe in the presence and absence of the respective unlabelled antigen and the binding data were converted into Scatchard plots (Scatchard, 1949). As shown in Table 2, affinity constants of this a/s for all the four glycoprotein hormones are nearly the same although the affinity for hLH was relatively higher compared to the other hormones. The affinity for hCGcY was much higher compared to all the glycoprotein hormones. Displacement of ‘2sI-labelled hormones by other hormones The hCGa a/s was incubated with ‘251-labelled hCG/hLH/hFSH in the presence of unlabelled hCG, hLH, hFSH and hCGa and binding of the labelled antigen to the a/s at a dilution of l/50,000 was determined. As shown in Fig. 2, each of these iodinated probes could be completely displaced by all other antigens suggesting that the antigenic determinants are conserved in all the three glycoprotein hormones used in this experiment.
Binding of MAbs to iodinated probes MAbs against hCG were produced and characterized as described elsewhere (Dighe et al., 1990). Five MAbs specific for the cx-subunit, but having different epitope specificities, were selected for these studies. All the five MAbs could bind [‘251]hCG, hLH, hFSH and hTSH, as well as hCG a-subunit suggesting the presence of the epitope in these proteins (Dighe et al., 1990). To determine if these MAbs were conformation specific, hCGa was reduced and carboxymethylated and then radioiodinated (RCM hCGa). None of these MAbs could bind to the RCM hCGa suggesting the conformation-specific nature of these MAbs. In order to determine to what extent the epitope recognized by MAbs in hCG are conserved in other glycoprotein hormones, displacement analysis was carried out. MAbs were adsorbed on to a plastic tube through an immunochemical bridge as described elsewhere (Murthy et al., 1989). Briefly, plastic tubes were coated with normal mouse serum which was then saturated with antimouse IgG raised in the goat. The tubes were finally coated with MAb at appropriate concentrations. Each MAb (solid phased) was incubated with [‘*‘I]hCG in the presence of all the four hormones, the a-subunit as well as RCM hCG a-subunit and binding was determined after overnight incubation at room temperature. The binding data were analyzed with the four-parameter logistic equation program and the EC,o determined by the computer are shown in Table 3. It was found that RCM hCG a-subunit was unable to displace hCG from binding to any of the MAbs again confirming that these MAbs are conformation specific and do not recognize any sequence in the a-subunit. It was also clear that the displacement of the labelled hCG varied depending on the MAb and displacing agent. Thus, the epitope recognized by the MAb 52/8 was present in all the glycoprotein hormones and appears to be present in nearly the same conformation as demonstrated by identical EC,, for all the antigens except hTSH which showed a higher ECSo. The other epitope that appeared to be present in a nearly similar conformation was the epitope recognized by the MAb 52/21. The epitope recognized by MAb 52/2 seems to be more specific for hCG as shown by its relatively higher affinity for hCG. Similarly affin-
67
I-
3-
3-
61O-
O-
125
10 O-
I
hLH
I3o-
6 Os x
4O- -
cl0 \ m _ 2 0.
M
hCG
U-0
hLH
A-A
hFSH
O-O
hCG
\
ng disploclng
Fig. 2. Displacement of binding incubated with [ 125 I] hCG (Fig. overnight at room temperature determined by incubating each concentration of the unlabelled
\
alpha
agent
of [‘251]hCG/hLH/hFSH to hCGn a/s by hCGa, hCG, hLH and hFSH. hCGa a/s (l/50,000) was 2a), [‘251]hLH (Fig. 2b) and [ ‘251]hFSH (Fig. 2c) in the presence of hCG, hLH hFSH and hCGa and binding was determined as described in Materials and Methods. The nonspecific binding was labelled antigen with normal rabbit serum and was subtracted from the binding observed with each hormone. The binding observed in the absence of any unlabelled antigen was termed & and considered as 100%. The values are means of duplicates.
68
TABLE
3
ANALYSIS OF DISPLACEMENT OF BINDING OF [‘251]hCG TO MAbs BY GLYCOPROTEIN CENTRATION REQUIRED TO OBTAIN 50% DISPLACEMENT (PMOL* SE/TUBE)
HORMONES:
CON-
MAbs were adsorbed on to a plastic tube through an immunochemical bridge. Each MAb was incubated with [‘251]hCG in the presence of 10-1000 ng of each displacing agent and binding was determined after overnight incubation at room temperature. The binding data were analyzed on an Apple IIe computer using the four-parameter logistic equation. The numbers in parentheses indicate the 95% confidence limits of the EC,,. MAb
hCG
hCGa
hLH
hFSH
hTSH
52/2
2.44*0.2 (2.0-2.9) 1.55 f 0.1 (1.4-1.72) No dis.
No dis. a
No dis.
No dis.
No dis.
1.48+0.1 (1.32-1.72) 0.9*0.1 (0.7-1.14) 8.45 f 0.8 (7-10.3) 10.96 + 0.25 (10.4-11.48)
2.2*0.16 (1.9-2.56) 13.7kO.8 (12.2-15.4) 2.4 + 0.2 (2.07-2.83) 5.5 +0.14 (5.2-5.8)
1.5*0.1 (1.38-1.76) 14.7*1.2 (12.3-17.5) 5.1 * 0.4 (4.38-6) 30.5 + 1.83 (26.9-34.5)
8.14kO.43 (8.14-9.08) No dis.
52/8 52/11 52/21 G,,,/f,
3.9 + 0.27 (3.34-4.46) 0.76 f 0.04 (0.69-0.94)
a No dis. = less than 50% displacement hCGa.
obtained
with 1 ng_ of the displacing
ity of MAb G,,/f, was significantly higher for hCG than that for hLH, hFSH and hCG&. The affinity of MAb 52/11 was surprisingly higher for the isolated subunit than for hLH and hFSH which in turn was much higher than that for hCG. Discussion It is known that the subunits of glycoprotein hormones undergo considerable conformational change when they combine to form the native, biologically active hormone (Ajoj et al., 1973; Ingham et al., 1973; Bewley et al., 1974; Bewley, 1978). However, these changes were of a gross nature as shown by changes in the circular dichroism spectrum or increase in the binding of anilino-naphtho-sulfonic acid to the hormone and did not yield any information about changes in the specific regions of the hormone molecule. In this study we investigated changes in the a-subunit of glycoprotein hormones when it is in combination with various P-subunits of the glycoprotein hormones using polyclonal and monoclonal antibodies as the probes. The polyclonal antiserum used in this study is highly specific for the a-subunit and had no cross-reactivity with hCG P-subunit. The cu-subunit was found to undergo conformational change
agent.
No displacement
35.6 +4 (28.16-45) No dis.
was obserued with
I pg RCM
when in combination with the P-subunit. This was shown by demonstrating shifts in the displacement curve. The shift depended on the P-subunit with which the a-subunit was present. Thus, while with hLH/I the a-subunit seemed to have undergone the least change, it has undergone relatively more changes when the P-subunit was either of FSH or TSH. However, the fact that this a/s binds to the a-subunit in all the four a-/3 dimers with nearly the same affinity suggests that the conformation of the a-subunit in all the hormones must be nearly similar. The proof for this possibility is provided by the observation that it was possible to completely displace binding of [ 1251]hCG/ hLH/ hFSH by either of these agents even at lower dilutions of the a/s. These results again imply that conformation of the a-subunit in the four human hormones must be very similar. However, the above results point out only gross similarities in the conformation of the a-subunit in the glycoprotein hormones. Hence these were followed by studies with MAbs. The studies carried out with the MAbs have revealed several interesting features. The MAbs used in this study were raised against hCG, but were specific for the a-subunit of hCG. These MAbs recognized unique conformation of the antigen and did not recognize any specific sequences as indicated by absence of
69
binding of RCMa to any of the MAbs. All these MAbs recognized and could bind ‘251-labelled human glycoprotein hormones and the a-subunit. This showed the existence of the common epitopes in all the four glycoprotein hormones. Displacement analysis with all the five MAbs using [‘251]hCG as the tracer showed that there are distinct similarities in many of the epitopes. The epitope recognized by MAb 52/8 appears to be identical in all the four giycoprotein hormones since hCG, hFSH, hLH and hCGa: were able to displace [‘25]hCG with equal efficacy. Thus this epitope is expressed in the a-subunit but is not altered as a result of recombination with any of the P-subunits. The epitope 52/21 was also conserved in all the antigens, although it was modified as indicated by significant shifts in the EC,,. The epitope recognized by Mab G,,/f, is similarly conserved in hCG, hLH, hFSH and hCG&, but its affinity for hCG was much higher than that for other hormones. The affinity of MAb 52/2 for hCG appeared to be very high for hCG relative to other hormones as no displacement with any other hormone could be observed suggesting that this epitope is altered in other a-@ dimers. However, it must be pointed out that all the four glycoprotein hormones and the a-subunit bind to all the MAbs (Dighe et al., 1990) suggesting that the epitope is present in all the four hormones although with slightly altered conformation. The most surprising results were obtained with MAbs 52/11. This MAb seems to have very high affinity for hCGa, hLH and hFSH than that for hCG. The reasons for this result are not presently known, but a different MAb recognizing the epitope very close to that recognized by this MAb also shows similar displacement curves (data not shown). The above results show that there are gross similarities in the conformation of the a-subunits in the four glycoprotein hormones. This is demonstrated by the use of polyclonal antibodies. Similarly many epitopes are conserved in all the four glycoprotein hormones. This is particularly true of the epitopes recognized by MAbs 52/8 and 52/21. However, there are dissimilarities also as indicated by shifts in the displacement curves of other MAbs. The dissimilarities in epitopes have been reported by Hojo and Ryan (1985) with two monoclonal
antibodies raised against hFSH but also capable of binding other glycoprotein hormones. However, both these MAbs appear to be recognizing the same epitope as their characteristics are nearly the same. In contrast, we have five MAbs with different characteristics as shown by different displacement curves, as well as binding characteristics (Dighe et al., 1990). Similar conformations of the a-subunit suggest several interesting possibilities. The four glycoprotein hormones have an identical a-subunit. There are a large number of reports indicating that the integrity of the a-subunit is absolutely essential for expression of biological activity of the hormones (see Sairam, 1983). The similar nature of the epitopes in the a-subunit in all glycoprotein hormones further underscores the importance of the a-subunit. It is tempting to speculate that the conserved conformation of the epitopes in the a-subunit of the native hormones plays an important role in the hormone action, viz. stimulation of G proteins and activation of adenylyl cyclase in the respective target organs, which is a common mechanism of hormone action. It can be hypothesized that the primary role of the hormone-specific P-subunit is to recognize the receptor and bring the a-subunit in proper orientation so that it can interact with regions of the receptor which are involved in signal transduction. In other words, the information necessary to stimulate the G proteins resides in the a-subunit. Our earlier studies with hCG and ovine FSH have clearly shown that the initial interaction between the hormones and their receptors takes place initially through the ,&subunits (Dighe and Moudgal, 1983; Moudgal et al., 1989). Although the inability of the a-specific MAbs as well as polyclonal antibodies to bind to receptor-bound hormone has been reported (Moyle et al., 1982; Milius et al., 1983), these experiments were carried out with a previously formed hormone-receptor complex and the time course of binding was never investigated as carried out by us (Dighe and Moudgal, 1983). Thus, the role of the P-subunit is probably to deliver the a-subunit, which by itself cannot bind to the receptor (Dighe et al., 1979), to the receptor site which, in turn, stimulates the response in the target organ. The ability of synthetic peptides from the a-subunit to stimulate the steroidogenic response is in total agreement with this concept
70
(Ryan et al., 1987). The recent demonstration of the importance of the glycosylation site at asparagine 52 (Matzuk et al., 1989) and involvement of histidine residues (Willey and Leidenberger, 1989) of the a-subunit in stimulation of response further underscores the importance of the a-subunit in hormone action. The precise localization of the epitopes in the a-subunit would certainly help in understanding the signal transduction mechanism. Several attempts have been made in the literature to locate these epitopes using synthetic peptides (Bidart et al., 1987, 1988, 1989; Troalen et al., 1988). We have already located some of these epitopes in the hCG molecule (Murthy et al., manuscripts in preparation). We also have observed that the MAbs against hCG &subunit are relatively more effective blockers of hormone-receptor interaction than the aspecific MAbs (Dighe, unpublished observations) which again indicates the role of P-subunit in binding of the hormone to the receptor. Another interesting point that can be highlighted from these studies is that although the P-subunits of hCG and hLH are very similar and both bind to the same receptor, there are subtle differences in the folding of the a-subunit in these dimers. This may perhaps explain the relatively higher potency as well as affinity of hCG for its receptors compared to other LHs (Sheela Rani and Moudgal, 1985). Acknowledgements
The work reported here was supported by grants provided by the Department of Biotechnology, Government of India and the Indian Council of Medical Research, New Dehli. References Ajoj, S.M., Edlehoch, H., Ingham, K.C., Morgan, F.C.. Canfield, R.E. and Ross, G.T. (1973) Arch. Biochem. Biophys. 159,497-504.
Bewley, T.A. (1979) Recent Prog. Horm. Res. 35, 135-213. Bewley, T.A., Sairam, M.R. and Li. C.H. (1974) Arch. Biothem. Biophys. 163, 625. Bidart, J.M., Troalen, F., Bohuon, C., Hennen, G. and Bellet, D.H. (1987) J. Biol. Chem. 262,15483-15489. Bidart, J.M., Troalen, F., Bousfield, G.R., Biekrn, S. and Bellet, D.H. (1988) J. Biol. Chem. 263, 10364-10369. Bidart, J.M., Troalen, F., Bousfield, G.R., Bohuon, C. and Bellet, D.H. (1989) Endocrinology 124, Y23-989. Diphe, R.R. and Moudgal, N.R. (1981) in Functional Correlates of Hormone Receptors in Reproduction (Mahesh, V.B., Maldoon, T.G., Saxena, B.B. and Sadler, W.A., eds.), pp. 545-549, Eisevier/No~h-Holland, Amsterdam. Digbe, R.R. and Moudgal, N.R. (1983) Arch. Biochem. Biophys. 225, 490-499. Dighe, R.R., Muralidhar, K. and Moudgal, N.R. (1979) Biothem. J. 180, 573-580. Dighe, R.R., Murthy, G.S. and Moudgal, N.R. (1990) J. Immunol. Methods (in press). Hojo, H. and Ryan, R.J. (1985) Endocrinology 117,2428-2434. Ingham, K.C., Ajoj. SM. and Edelhoch, M. (1973) Arch. Biochem. Biophys. 159, 596-605. Matzuk, M.M., Keene, J.L. and Boime, I. (1989) J. Biol. Chem. 264, 2409-2414. Milius, R.P., Midgeley, A.R. and Birken, S. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 7375-7378. Moudgal, N.R., Sairam. M.R. and Dighe, R.R. (1989) J. Biosci. 14,91-100. Moyle, W.R., Ehrlich, P.H. and Canfield, R.E. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2245-2249. Murthy, G.S., Laxmi, B.S. and Moudgal, N.R. (1989) J. Biosci. 14, 9-20. Pierce, J.G. and Parsons, T.F. (1981) Annu. Rev. Biochem. 50, 465. Ryan, R.J., Keutmann, H.T., Charlsworth. M.C.. McCormick, D.J., Milius, R.P., Calvo, F.O. and Vutyavanich, T. (1987) Recent Prog. Horm. Res. 47, 383-429. Sairam, M.R. (1983) in Hormones and Hormonal Peptides. Vol. 11 (Li, Ch., ed.), pp. l-79. Academic Press, New York. Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 51,660-662. Sheela Ram, C.S. and Moudgal, N.R. (1985) Endocrinology, 116, 597-603. Troalen, F., Bellet, D.H., Ghillani, P., Puisieux, A., Bohuon, C.J. and Bidart, J.M. (1988) J. Biol. Chem. 263, 10370-10376. Willey, K.P. and Leidenberger, F. (1989) J. Biol. Chem. 264. 19716-19729.
