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Some characteristics of GnRH receptors in rat-pituitary membranes: Differences between an agonist and an antagonist
Molecular and CellularEndocrinology, 23 ( 198 1) 215 -28 1 Elsevier/North-Holland Scientific Publishers, Ltd.
215
SOME CHARACTERISTICS OF GnRH RECEPTORS IN RAT-PITUITARY MEMBRANES: DIFFERENCES BETWEEN AN AGONIST AND AN ANTAGONIST Eli HAZUM Department of Hormone Research, The WeizmannInstitute of Science, Rehovot, P.O. Box 26, 76100 (Israel) Received 2 March 1981; accepted 9 May 1981
The interaction of gonadotropin-releasing hormone (GnRH) agonists and antagonists with pituitary membranes was studied by using t 2 5 I-labeled agonist, [ D-Ser(t-Bu)6, des-Gly 1Oethylamide] GnRH, and antagonist [ D-pGlttl , D-Phe2, Trp’ ‘6]-GnRH. Their binding was affected differently by cations, and by pretreatment of membranes with proteolytic enzymes and sulfhydryl-blocking reagents. Monovalent cations at millimolar concentrations (lo-100 mM) and divalent cations at lower concentrations (0.54 mM) reduced more significantly the binding of the agonist than that of the antagonist. Pretreatment of the membranes with trypsin and chymotrypsin abolished the specific bindii of both agonist and antagonist, in a doseresponse manner, with the former being less affected. Pretreatment of the membranes with sulfhydryl-blocking reagents did not alter the binding of the antagonist but enhanced the binding of the agonist. This enhancement in the specific binding was found to be due to an increase in the apparent affinity of the agonist. These results may suggest that GnRH agonists and antagonists bind differently to the same receptor. Keywords: adenohypophysis;
hormone receptors; GnRH agonist and antagonist binding.
The first step in GnRH action is its recognition by specific binding sites (receptors) at the surface of gonadotrope cells (Vale et al., 1976). The interaction of GnRH with pituitary membrane preparations or pituitary cells has been studied in detail by using radio-iodinated, metabolically stable GnRH analogs (Clayton et al., 1978, 1979; Conne et al., 1979; Naor et al., 1980; Hazum et al., 1980; Meidan and Koch, 1981). These studies have indicated the presence of a single class of highaffinity binding sites for both agonists and antagonists of GnRH.. Although these high-affinity binding sites probably represent physiological receptors for GnRH, the differences between agonists and antagonists binding has not been studied. Here I report on the characterization and properties of the GnRH receptors in pituitary
Abbreviation: GnRH, gonadotropin-releasing 0303-7207/81/0000-0000/$02.50
hormone.
0 1981 Elsevier/North-Holland
Scientific Publishers, Ltd.
276
E. Hazum
membranes and the differential effects of cations, proteolytic enzymes and sulfhydryl-disulfide reagents on the binding of GnRH agonists and antagonists.
MATERIALS Dithiothreitol, iodoacetate, iodoacetamide and N-ethylmaleimide were purchased from Sigma. Trypsin (130 U/mg) and chymotrypsin (49.2 U/mg) were purchased from Worthington and Millipore Corporation, respectively.
METHODS Iodination and pituitary membrane preparations
[D-Ser/(t-Bu)6, des-Glyloethylamide]-GnRH (Buserelin, provided by Dr. J. Sandow, Hoechst, Frankfurt) and [D-pG1~' , D-Phe2, Trp3 y6]-G&H (kindly supplied by Drs. W. Vale and J. Rivier, Salk Institute, La Jolla, CA) were iodinated by the lactoperoxidase method, applied to a column of Sephadex G-25 (4 cm X 35 cm) previously equilibrated and eluted with 0.01 M acetic acid (Sandow and Konig, 1979). The specific activity of the labeled peptides was about 1500 &i/lg. Pituitary membranes were prepared from Wistar-derived female rats according to Herber et al. (Herber and Odell, 1978) with modifications. Briefly, the glands were homogenized gently with a Dounce homogenizer at 4°C in 1 mM NaHC03 containing 1 mM dithiothreitol and centrifuged for 10 min at 1000 X g. The supematant was then centrifuged for 20 min at 20 000 X g. The pellet was resuspended in assay buffer (10 mM Tris-HCI, pH 7.4, containing 0.1% bovine serum albumin, BSA), centrifuged at 20 000 X g for 20 min and finally suspended in assay buffer. Binding assay
The labeled peptides (40 000 cpm) were incubated with 150-300 c(gprotein of pituitary membranes in a total volume of 0.5 ml assay buffer for 90 min at 4°C (equilibrium conditions). The homogenate was then filtered under vacuum through Whatman GF/C filters, presoaked in 0.1% of BSA, washed with 10 ml of ice-cold incubation buffer and the filters were counted in a y-counter. Specific binding represents the bound radioactivity that can be displaced by prior addition of 10m6 M unlabeled Buserelin or [D-pG1~' , D-Phe2, Trp3p6]GnRH. Each value is the mean of duplicate incubations, which varied by less than 7%. RESULTS AND DISCUSSION Both [r2’I]-Buserelin (GnRH agonist) and [12’1] [D-pGl~‘, D-Phe’, Trp3*6]GnRH (GnRH antagonist) exhibited high binding affinity to pituitary membrane
Differential binding of GnRH agonistsand antagonists
211
preparations; the Kd values were 0.5 and 0.04 nM, respectively (Figs. 1 and 2, control membranes). The inhibition of the binding of [1251]Buserelin by the antagonist [D-pGlu’, D-Phe’, Trp3v6]-GnRH and the inhibition of the binding of the 1251-labeledantagonist by the agonist (Figs. 1 and 2) indicate that both can interact with the same receptors. The effects of cations on the binding of [’ “11 Buserelin compared with [’ “11 [D-pG1~' , D-Phe2, Trp3y6]-GnRH are given in Table 1. Monovalent cations, Na’ and K’, decreased substantially the binding of 1251-labeledagonist (about 45% inhibition at 50 mM) but only slightly affected (20% inhibition at 50 mM) 1251-labeledantagonist binding. Divalent cations, Mg2+ and Ca’+, showed similar patterns in affecting the binding of ’ 25 I-labeled agonist and antagonist. However, the effect was at lower concentration (l-5 mM). Some of the cationic effects on agonists binding have been published previously (Clayton et al., 1978; Marian and Conn, 1980; Perrin et al., 1980) and are consistent with the results presented in this study. Guanosine-5’-triphosphate (GTP, 0.1 mM) slightly affected the binding of agonist and antagonist, 84% and 91%, respectively.
Fig. 1. Competition of binding of [ 1251]-Buserelin to control membranes (A- - - - - -A) and to by [D-pClul, D-Phe’, TIP~,~]membranes pretreated with 0.1 mM iodoacetate (* -) GnRH (A) and by Buserelin (B, inset, Scatchard plot). The radioactive Buserelin (40 000 cpm) was incubated with various concentrations of the tested compound for 90 min at 4°C in a final volume of 0.5 ml containing pituitary membranes (0.15-0.3 mg of protein/ml), and the bmding was measured as described in Materials and Methods and Table 1. Fig. 2. Competition of binding of [12SI][D-pClu1, D-Phe’, Trp3p6]-CnRH to control membranes (A- - - - - -A) and to membranes pretreated with 0.1 mM iodoacetate (0 -) by [DpGlu’ , D-Phe’, Trp3*6]-CnRH (A) and by Buserelin (B). The radioactive [D-pClur , D-Phe’, Trp3 8 1CnRH (40 000 cpm) was incubated as described in Fig. 1.
E. Hazum
278
Table 1 Differences in the ability of various cations and GTP to alter the binding of 12sI-labeled Buserelin compared with rssI-labeled [~pGlur , DPhes, Trp3Fe]GnRH Specific binding (%) a)
Cation (mM)
Control
[DpClur , DPhes, Trp3Fe]GnRH
[tasI]-Buserelin
[tssI]
1006b)
100+5 c)
Na+,
5 10 50 100
a9t4 79f5 55-55 34 + 1
102 k 8 86 f 2 8026 61_+2
K+,
10 50 100
87f4 54f2 46 + 3
85k4 8025 654
Mg2+,
0.5 1 5
79 + 2 71+2 46f5
98 k 2 93+5 63+8
Gas+,
0.5 1 5
74_+7 51+4 42 k 3
86-e5 68 f 3 60 f 2
GTP,
0.1
84 _+8
9129
a) Specific binding was deter&ted as described under Materials and Methods. This experiment was repeated twice (in duplicate) and the results are expressed as percentage of the control + S.E. b) 100% = 6.8 fentomoles/mg protein, or 6% specific binding of total. c) 100% = 18.1 fentomoles/mg protein, or 16% specific binding of total.
The effects of trypsin and chymotrypsin on the binding of ’ 25I-labeled Buserelin and [D-pG1~’ , D-Phe2, Trp3y6]-GnRH are shown in Table 2. Incubation of the membranes (20 min at 37°C without the enzymes) resulted in about 40% inhibition of the specific binding. Pretreatments of the membranes with both trypsin (0.1-100 pg/ml) and chymotrypsin (0.1-100 fig/ml) resulted in a decrease of the specific binding of 1251-labeledagonist and antagonist in a dose-response manner. However, [12’1] [D-pGlu’, D-Phe’, Trp3s6]-GnRH binding was more sensitive to both enzymes compared with [’ 251]-Buserelin binding. The effects of sulthydryl and disulfide reagents on the binding of 12SI-labeled Buserelin and [D-pGlu’, D-tie’, Trp3j”]-GnRH are shown in Table 3. Dithiothreito1 and sulfhydryl blocking reagents (Nethylmaleimide, iodoacetamide and iodoacetate) did not alter significantly the binding of the 1251-labeledantagonist. Similarly, dithiothreitol did not affect the binding of the agonist. However, pretreatment of the membranes with sulfhydryl-blocking reagents (0.01-l mM) enhanced the binding of [1251]-Buserelin. To further examine these effects, the competition
Differential binding of GnRH agonistsand antagonists Table 2 Effect of trypsin and chymotrypsin on (12SI]Buserelin and [‘*‘I] GnRH binding to pituitary membranes Condition a)
219
[D-pGlu’, D-Phe*, Trp3*‘j]-
Specific binding (%) b)
Control (4°C) 20 min at 37°C
[tZ51]Buserelin
[‘25~][D-~lu’,
loot8 58+3
lOOk7 61&S
Trypsin,
0.1 1 10 100
pg/ml rg/ml Ccg/ml &ml
56+4 39 t 3 824 0
39k4 25 k 3 2t1 0
Chymotrypsin,
0.1 1 10 100
Mg/ml rcg/ml &ml &ml
57+5 45+4 24 I! 3 3+1
47 t 6 27?3 7_+2 0
D-Phe*, Trp3,6 ]GnRH
a) The effect of enzymes was tested by incubating (20 min at 37’?I) various concentrations of trypsin or chymotrypsin with pituitary membranes in Tris-HCl buffer, pH 7.4. The reaction was terminated by washing (in assay buffer) and centrifugation (twice). b) Specific binding was determined as described in Materials and Methods and Table 1.
of binding of ‘*‘I-labeled Buserelin (Fig. 1) or 12SI-labeled [D-pGlur, D-me*, Trp3y6]-GnRH (Fig. 2) by Buserelin and by [D-pG1~’ , D-me*, Trp3y6]-GnRh, to control membranes-and to membranes pretreated with 0.1 mM iodoacetate, were studied. The apparent IC,, values (the concentration of unlabeled ligand at which the maximal specific binding of labeled ligand is displaced by 50%) for the binding of the antagonist (Fig. 1A) were 0.04 and 0.03 nM for control and pretreated membranes, respectively. The apparent Kd values for Buserelin (Fig. 1B) were 0.5 and 0.12 nM (Fig. lB, Scatchard plot) for control and pretreated membranes, respectively. This suggests that the enhanced binding (after pretreatment with sulfhydrylblocking reagents) is mainly due, to an increase of the apparent affinity of the agonist. As shown in Fig. 2A and B, there were no significant changes in the competition of binding of 1251-labeled [D-pGl~‘, D-me*, Trp3p6]-GnFW to control and pretreated membranes by the agonist and the antagonist. The apparent IC,, values (Fig. 2, control membranes) were 0.12 and 0.06 nM for Buserelin and [D-pGl~*, D-Phe*, Trp3y6]-GnRH, respectively. These results may suggest that the effect of sulfhydryl-blocking reagents is due to the presence of free sulfhydryl groups near the binding sites of the hormone, which upon blockage increases agonist binding without altering antagonist binding. In summary, these studies indicate that GnRH agonists and antagonists can interact with the same receptor, despite their different biological actions. However, the binding of agonists and antagonists differs with respect to the effect of ions, proteo-
E. Hazum
280
Table 3 Effect of sulfhydryl and disulfide reagents on the binding of [ 1251]Buserelin and [ 1251][ DpClur , D-Phe2, Trpsle]GnRH Reagent a) (mM)
Specific binding (% control) b) [rssI]-Buserelin
[rssI]
N-Ethylmaleimide 0.01 0.1 1
111+17 125 + 28 131222
107+ 2* 97 t 10 94513
Iodoacetamide 0.01 0.1 1
llO& 5 130?25 153*17*
105 + 4 122+ 3** 105 5 2
Iodoacetate 0.01 0.1 1
123+ 9* 142+16* 165 + 20 *
109+ 1175 96:
Dithiothreitol 0.1 1
92+ 8 98+12
[D-pGlur, D-Phes, Trp3p6]GnRH
1 ** 2** 3
102 _+10 107t12
a) AU reagents were dissolyed immediately before use. When N-ethylmaleimide, iodoacetamide and iodoacetate were tested the pituitary membranes were pretreated (in Tris-HCl buffer) with the reagent for 45 min at 4”C, washed twice (in assay buffer) by centrifugation and the binding was measured as described in Materials and Methods and Table 1. b) This experiment was repeated 4 times owing to high variability. The results are expressed as percentage of the control + S.E. Values designated by * and ** are significantly different from control at P < 0.05 and 0.001, respectively, according to Student’s f test.
lytic enzymes and sulthydryl-blocking reagents. This suggests that GnRH agonists and antagonists bind differently to the same receptor or may also be related to possible conformational changes or steric-allosteric effects of the receptor or the hormone. ACKNOWLEDGEMENTS This work was supported by the Ford Foundation and the Rockefeller Foundation, New York. REFERENCES Clayton, R.N., Shakespear, R.A., and MarshaIl, J.C. (1978) Mol. Cell. Endocrinol. 11,63-78. Clayton, R.N., Shakespear, R.A., Duncan, J.A., and Marshall, J.C. (1979) Endocrinology 105, 1369-1376.
Differential binding of GnRH agonists and antagonists
281
Conne, B.S., Aubert, M.L., and Sizonenko, PC. (1979) Biochem. Biophys. Res. Commun. 90, 1249-1256. Hazum, E., Cuatrecasas, P., Marian, J., and Conn, P.M. (1980) Proc. Natl. Acad. Sci. (U.S.A.) 77,6692-6695. Herber, D., and Odell, W.D. (1978) Biochem. Biophys. Res. Commun. 82,67-73. Marian, J., and Conn, P.M. (1980) Life Sci. 27,87-92. Meidan, R., and Koch, Y. (1981) Life Sci., in press. Naor, Z., Clayton, R.N., and Catt, K.J. (1980) Endocrinology 107, 1144-l 152. Perrin, M.H., Rivier, J.E., and Vale, W.W. (1980) Endocrinology 106, 1289-1296. Sandow, J., and Konig, W. (1979) J. Endocrinol. 81,175-182. Vale, W., Rivier, C., Brown, M., Leppahroto, J., Ling, N., Monahan, M., and Rivier, J. (1976) Clin. Endocrinol. 5 (Suppl.), 2615-2735.
215
SOME CHARACTERISTICS OF GnRH RECEPTORS IN RAT-PITUITARY MEMBRANES: DIFFERENCES BETWEEN AN AGONIST AND AN ANTAGONIST Eli HAZUM Department of Hormone Research, The WeizmannInstitute of Science, Rehovot, P.O. Box 26, 76100 (Israel) Received 2 March 1981; accepted 9 May 1981
The interaction of gonadotropin-releasing hormone (GnRH) agonists and antagonists with pituitary membranes was studied by using t 2 5 I-labeled agonist, [ D-Ser(t-Bu)6, des-Gly 1Oethylamide] GnRH, and antagonist [ D-pGlttl , D-Phe2, Trp’ ‘6]-GnRH. Their binding was affected differently by cations, and by pretreatment of membranes with proteolytic enzymes and sulfhydryl-blocking reagents. Monovalent cations at millimolar concentrations (lo-100 mM) and divalent cations at lower concentrations (0.54 mM) reduced more significantly the binding of the agonist than that of the antagonist. Pretreatment of the membranes with trypsin and chymotrypsin abolished the specific bindii of both agonist and antagonist, in a doseresponse manner, with the former being less affected. Pretreatment of the membranes with sulfhydryl-blocking reagents did not alter the binding of the antagonist but enhanced the binding of the agonist. This enhancement in the specific binding was found to be due to an increase in the apparent affinity of the agonist. These results may suggest that GnRH agonists and antagonists bind differently to the same receptor. Keywords: adenohypophysis;
hormone receptors; GnRH agonist and antagonist binding.
The first step in GnRH action is its recognition by specific binding sites (receptors) at the surface of gonadotrope cells (Vale et al., 1976). The interaction of GnRH with pituitary membrane preparations or pituitary cells has been studied in detail by using radio-iodinated, metabolically stable GnRH analogs (Clayton et al., 1978, 1979; Conne et al., 1979; Naor et al., 1980; Hazum et al., 1980; Meidan and Koch, 1981). These studies have indicated the presence of a single class of highaffinity binding sites for both agonists and antagonists of GnRH.. Although these high-affinity binding sites probably represent physiological receptors for GnRH, the differences between agonists and antagonists binding has not been studied. Here I report on the characterization and properties of the GnRH receptors in pituitary
Abbreviation: GnRH, gonadotropin-releasing 0303-7207/81/0000-0000/$02.50
hormone.
0 1981 Elsevier/North-Holland
Scientific Publishers, Ltd.
276
E. Hazum
membranes and the differential effects of cations, proteolytic enzymes and sulfhydryl-disulfide reagents on the binding of GnRH agonists and antagonists.
MATERIALS Dithiothreitol, iodoacetate, iodoacetamide and N-ethylmaleimide were purchased from Sigma. Trypsin (130 U/mg) and chymotrypsin (49.2 U/mg) were purchased from Worthington and Millipore Corporation, respectively.
METHODS Iodination and pituitary membrane preparations
[D-Ser/(t-Bu)6, des-Glyloethylamide]-GnRH (Buserelin, provided by Dr. J. Sandow, Hoechst, Frankfurt) and [D-pG1~' , D-Phe2, Trp3 y6]-G&H (kindly supplied by Drs. W. Vale and J. Rivier, Salk Institute, La Jolla, CA) were iodinated by the lactoperoxidase method, applied to a column of Sephadex G-25 (4 cm X 35 cm) previously equilibrated and eluted with 0.01 M acetic acid (Sandow and Konig, 1979). The specific activity of the labeled peptides was about 1500 &i/lg. Pituitary membranes were prepared from Wistar-derived female rats according to Herber et al. (Herber and Odell, 1978) with modifications. Briefly, the glands were homogenized gently with a Dounce homogenizer at 4°C in 1 mM NaHC03 containing 1 mM dithiothreitol and centrifuged for 10 min at 1000 X g. The supematant was then centrifuged for 20 min at 20 000 X g. The pellet was resuspended in assay buffer (10 mM Tris-HCI, pH 7.4, containing 0.1% bovine serum albumin, BSA), centrifuged at 20 000 X g for 20 min and finally suspended in assay buffer. Binding assay
The labeled peptides (40 000 cpm) were incubated with 150-300 c(gprotein of pituitary membranes in a total volume of 0.5 ml assay buffer for 90 min at 4°C (equilibrium conditions). The homogenate was then filtered under vacuum through Whatman GF/C filters, presoaked in 0.1% of BSA, washed with 10 ml of ice-cold incubation buffer and the filters were counted in a y-counter. Specific binding represents the bound radioactivity that can be displaced by prior addition of 10m6 M unlabeled Buserelin or [D-pG1~' , D-Phe2, Trp3p6]GnRH. Each value is the mean of duplicate incubations, which varied by less than 7%. RESULTS AND DISCUSSION Both [r2’I]-Buserelin (GnRH agonist) and [12’1] [D-pGl~‘, D-Phe’, Trp3*6]GnRH (GnRH antagonist) exhibited high binding affinity to pituitary membrane
Differential binding of GnRH agonistsand antagonists
211
preparations; the Kd values were 0.5 and 0.04 nM, respectively (Figs. 1 and 2, control membranes). The inhibition of the binding of [1251]Buserelin by the antagonist [D-pGlu’, D-Phe’, Trp3v6]-GnRH and the inhibition of the binding of the 1251-labeledantagonist by the agonist (Figs. 1 and 2) indicate that both can interact with the same receptors. The effects of cations on the binding of [’ “11 Buserelin compared with [’ “11 [D-pG1~' , D-Phe2, Trp3y6]-GnRH are given in Table 1. Monovalent cations, Na’ and K’, decreased substantially the binding of 1251-labeledagonist (about 45% inhibition at 50 mM) but only slightly affected (20% inhibition at 50 mM) 1251-labeledantagonist binding. Divalent cations, Mg2+ and Ca’+, showed similar patterns in affecting the binding of ’ 25 I-labeled agonist and antagonist. However, the effect was at lower concentration (l-5 mM). Some of the cationic effects on agonists binding have been published previously (Clayton et al., 1978; Marian and Conn, 1980; Perrin et al., 1980) and are consistent with the results presented in this study. Guanosine-5’-triphosphate (GTP, 0.1 mM) slightly affected the binding of agonist and antagonist, 84% and 91%, respectively.
Fig. 1. Competition of binding of [ 1251]-Buserelin to control membranes (A- - - - - -A) and to by [D-pClul, D-Phe’, TIP~,~]membranes pretreated with 0.1 mM iodoacetate (* -) GnRH (A) and by Buserelin (B, inset, Scatchard plot). The radioactive Buserelin (40 000 cpm) was incubated with various concentrations of the tested compound for 90 min at 4°C in a final volume of 0.5 ml containing pituitary membranes (0.15-0.3 mg of protein/ml), and the bmding was measured as described in Materials and Methods and Table 1. Fig. 2. Competition of binding of [12SI][D-pClu1, D-Phe’, Trp3p6]-CnRH to control membranes (A- - - - - -A) and to membranes pretreated with 0.1 mM iodoacetate (0 -) by [DpGlu’ , D-Phe’, Trp3*6]-CnRH (A) and by Buserelin (B). The radioactive [D-pClur , D-Phe’, Trp3 8 1CnRH (40 000 cpm) was incubated as described in Fig. 1.
E. Hazum
278
Table 1 Differences in the ability of various cations and GTP to alter the binding of 12sI-labeled Buserelin compared with rssI-labeled [~pGlur , DPhes, Trp3Fe]GnRH Specific binding (%) a)
Cation (mM)
Control
[DpClur , DPhes, Trp3Fe]GnRH
[tasI]-Buserelin
[tssI]
1006b)
100+5 c)
Na+,
5 10 50 100
a9t4 79f5 55-55 34 + 1
102 k 8 86 f 2 8026 61_+2
K+,
10 50 100
87f4 54f2 46 + 3
85k4 8025 654
Mg2+,
0.5 1 5
79 + 2 71+2 46f5
98 k 2 93+5 63+8
Gas+,
0.5 1 5
74_+7 51+4 42 k 3
86-e5 68 f 3 60 f 2
GTP,
0.1
84 _+8
9129
a) Specific binding was deter&ted as described under Materials and Methods. This experiment was repeated twice (in duplicate) and the results are expressed as percentage of the control + S.E. b) 100% = 6.8 fentomoles/mg protein, or 6% specific binding of total. c) 100% = 18.1 fentomoles/mg protein, or 16% specific binding of total.
The effects of trypsin and chymotrypsin on the binding of ’ 25I-labeled Buserelin and [D-pG1~’ , D-Phe2, Trp3y6]-GnRH are shown in Table 2. Incubation of the membranes (20 min at 37°C without the enzymes) resulted in about 40% inhibition of the specific binding. Pretreatments of the membranes with both trypsin (0.1-100 pg/ml) and chymotrypsin (0.1-100 fig/ml) resulted in a decrease of the specific binding of 1251-labeledagonist and antagonist in a dose-response manner. However, [12’1] [D-pGlu’, D-Phe’, Trp3s6]-GnRH binding was more sensitive to both enzymes compared with [’ 251]-Buserelin binding. The effects of sulthydryl and disulfide reagents on the binding of 12SI-labeled Buserelin and [D-pGlu’, D-tie’, Trp3j”]-GnRH are shown in Table 3. Dithiothreito1 and sulfhydryl blocking reagents (Nethylmaleimide, iodoacetamide and iodoacetate) did not alter significantly the binding of the 1251-labeledantagonist. Similarly, dithiothreitol did not affect the binding of the agonist. However, pretreatment of the membranes with sulfhydryl-blocking reagents (0.01-l mM) enhanced the binding of [1251]-Buserelin. To further examine these effects, the competition
Differential binding of GnRH agonistsand antagonists Table 2 Effect of trypsin and chymotrypsin on (12SI]Buserelin and [‘*‘I] GnRH binding to pituitary membranes Condition a)
219
[D-pGlu’, D-Phe*, Trp3*‘j]-
Specific binding (%) b)
Control (4°C) 20 min at 37°C
[tZ51]Buserelin
[‘25~][D-~lu’,
loot8 58+3
lOOk7 61&S
Trypsin,
0.1 1 10 100
pg/ml rg/ml Ccg/ml &ml
56+4 39 t 3 824 0
39k4 25 k 3 2t1 0
Chymotrypsin,
0.1 1 10 100
Mg/ml rcg/ml &ml &ml
57+5 45+4 24 I! 3 3+1
47 t 6 27?3 7_+2 0
D-Phe*, Trp3,6 ]GnRH
a) The effect of enzymes was tested by incubating (20 min at 37’?I) various concentrations of trypsin or chymotrypsin with pituitary membranes in Tris-HCl buffer, pH 7.4. The reaction was terminated by washing (in assay buffer) and centrifugation (twice). b) Specific binding was determined as described in Materials and Methods and Table 1.
of binding of ‘*‘I-labeled Buserelin (Fig. 1) or 12SI-labeled [D-pGlur, D-me*, Trp3y6]-GnRH (Fig. 2) by Buserelin and by [D-pG1~’ , D-me*, Trp3y6]-GnRh, to control membranes-and to membranes pretreated with 0.1 mM iodoacetate, were studied. The apparent IC,, values (the concentration of unlabeled ligand at which the maximal specific binding of labeled ligand is displaced by 50%) for the binding of the antagonist (Fig. 1A) were 0.04 and 0.03 nM for control and pretreated membranes, respectively. The apparent Kd values for Buserelin (Fig. 1B) were 0.5 and 0.12 nM (Fig. lB, Scatchard plot) for control and pretreated membranes, respectively. This suggests that the enhanced binding (after pretreatment with sulfhydrylblocking reagents) is mainly due, to an increase of the apparent affinity of the agonist. As shown in Fig. 2A and B, there were no significant changes in the competition of binding of 1251-labeled [D-pGl~‘, D-me*, Trp3p6]-GnFW to control and pretreated membranes by the agonist and the antagonist. The apparent IC,, values (Fig. 2, control membranes) were 0.12 and 0.06 nM for Buserelin and [D-pGl~*, D-Phe*, Trp3y6]-GnRH, respectively. These results may suggest that the effect of sulfhydryl-blocking reagents is due to the presence of free sulfhydryl groups near the binding sites of the hormone, which upon blockage increases agonist binding without altering antagonist binding. In summary, these studies indicate that GnRH agonists and antagonists can interact with the same receptor, despite their different biological actions. However, the binding of agonists and antagonists differs with respect to the effect of ions, proteo-
E. Hazum
280
Table 3 Effect of sulfhydryl and disulfide reagents on the binding of [ 1251]Buserelin and [ 1251][ DpClur , D-Phe2, Trpsle]GnRH Reagent a) (mM)
Specific binding (% control) b) [rssI]-Buserelin
[rssI]
N-Ethylmaleimide 0.01 0.1 1
111+17 125 + 28 131222
107+ 2* 97 t 10 94513
Iodoacetamide 0.01 0.1 1
llO& 5 130?25 153*17*
105 + 4 122+ 3** 105 5 2
Iodoacetate 0.01 0.1 1
123+ 9* 142+16* 165 + 20 *
109+ 1175 96:
Dithiothreitol 0.1 1
92+ 8 98+12
[D-pGlur, D-Phes, Trp3p6]GnRH
1 ** 2** 3
102 _+10 107t12
a) AU reagents were dissolyed immediately before use. When N-ethylmaleimide, iodoacetamide and iodoacetate were tested the pituitary membranes were pretreated (in Tris-HCl buffer) with the reagent for 45 min at 4”C, washed twice (in assay buffer) by centrifugation and the binding was measured as described in Materials and Methods and Table 1. b) This experiment was repeated 4 times owing to high variability. The results are expressed as percentage of the control + S.E. Values designated by * and ** are significantly different from control at P < 0.05 and 0.001, respectively, according to Student’s f test.
lytic enzymes and sulthydryl-blocking reagents. This suggests that GnRH agonists and antagonists bind differently to the same receptor or may also be related to possible conformational changes or steric-allosteric effects of the receptor or the hormone. ACKNOWLEDGEMENTS This work was supported by the Ford Foundation and the Rockefeller Foundation, New York. REFERENCES Clayton, R.N., Shakespear, R.A., and MarshaIl, J.C. (1978) Mol. Cell. Endocrinol. 11,63-78. Clayton, R.N., Shakespear, R.A., Duncan, J.A., and Marshall, J.C. (1979) Endocrinology 105, 1369-1376.
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