Properties of prostaglandin F2α receptors in bovine corpus luteum cell membranes

Molecular and Cellular Endocrinology,

0 Elsevier/North-Holland

6 (1976)

l-16

Biomedical Press

PROPERTIES OF PROSTAGLANDIN LUTEUM CELL MEMBRANES

Fzor RECEPTORS

IN BOVINE CORPUS

Ch.V. RAO Departments of Obstetrics-Gynecology Medicine, Louisville, Kentucky 40202,

and Biochemistry, U.S.A.

University of Louisville, School of

Received 22 January 1976; accepted 9 April 1976

The specific binding of 3H-labeled prostaglandin (PG) Fao to bovine corpus luteum cell membranes was a rapid (Kl = 1.1 X lo4 M-’ s-l) and reversible (K-1 = 3.3 X lob4 s-l) process at 22°C. The specific binding was also a saturable process exhibiting two classes of receptors with apparent dissociation constants (Kds) of 1.6 X 10”) M and 2.4 X 10-e M. The heterogenous nature of [3H]PGF2a! binding does not appear to be due to negative cooperativity but merely to represent the existence of two independent groups of receptor sites with discrete affinities. Free energy changes of +11.9 and + 10.3 Kcal/mol were calculated from the Kds of high and low affinity receptors, respectively. The binding of L3H]PGFao to the membranes was not accompanied by any detectable changes in receptor-bound or free [‘H] PGFz,. Addition of increasing amounts of unlabeled PGFao resulted in a dosedependent inhibition of [ 3H]PGFa, binding to the membranes, with complete inhibition occurring at low6 M. Other unlabeled PCs such as PGFr,, PGEz (S-fold), PGEr (120-fold), PGAr and PGBr (about lO,OOO-fold) were less effective when compared to unlabeled PGFzo in inhibiting [3H]PGF20: binding to the membranes. The metabolites of PGFz,, 15keto-PGF2, and 13,14dihydro-15 keto-PGF2, had loo-fold less affinity for PGFzo receptors. 15(S)15-MethylPGF2,, an analogue of PGF?,, had a fairly high affinity but lower than its parent molecule. Various unsaturated fatty acids, indomethacin and 7-oxa-13-prostynoic acid had 3,000-to lO,OOO-fold less affinities for PGFz~ receptors. Incubation of membranes with various enzymes revealed that PGFao receptor molecules are protein in nature which require membrane lipids and specific phospholipids for binding function. Among the various phospholipids used, sphingomyelin was found to be very effective in restoring the loss of [3H]PGF2(U binding in phospholipase C-treated membranes. N-Ethylmaleimide, but not other SH group alkylating agents inhibited binding. The binding was also inhibited by tetranitromethane, dinitrofluorobenzene and acetic anhydride. This suggested that tyrosyl, histidyl, tryptophan and ammo (any one or all of them) but not SH groups were involved in binding interaction. Keywords:

bovine corpora lutea; prostaglandin F2o receptors; cell membranes.

Outer cell membranes of bovine corpora lutea have been shown to contain specific and high affinity receptors for human choriogonadotropin-lutropin (hCG-LH) (Rao, 1973; Gospodarowicz, 1973; Haour and Saxena, 1974; Menon and Kiburz,

Ch. V. Rao

2

1974) prostaglandin (PC) Es (Rao, 1973; Kimball and Lauderdale, 1975) and for PGFZ, (Rao, 1975a; Powell et al., 1975a; Kimball and Lauderdale, 1975). There are several reports describing in detail the properties of gonadotropin and PGE receptors in bovine corpora lutea (Gospodarowicz, 1973; Rao, 1974a,b; Haour and Saxena, 1974). Although there is some information on properties of PGFZa receptors (Rao, 1975a; Powell et al., 1975; Kimball and Lauderdale, 1975) it has not been as extensive as in the case of the other two receptors. Therefore, these studies were undertaken to explore further into the properties of PGF2, receptors.

EXPERIMENTAL

PROCEDURE

Materials Unlabeled PCs (natural, metabolites and an analogue) were generously donated by Dr. John Pike of the Upjohn Co., Kalamazoo, Mich. The 7-oxa-13-prostynoic acid was kindly supplied by Dr. F.A. Kuehl, Jr., of the Merck Institute for Therapeutic Research, Rahway, N.J. Unlabeled hCG (10,600 IU/mg) was a gift from the Center for Population Research, National Institutes of Child Health and Human Development, Bethesda, Md. The following chemicals were purchased from the commercial sources indicated: [3H]PGF2a, (178 Ci/mmol) from New England Nuclear Corp.; phospholipase A (Crotalus terrificus terrificus, 850.0 units/mg), phospholipase C (0. welchii, 2.0 IU/mg), phospholipase D (0.53 units/mg), lipase (81.0 units/mg), pronase (73 .O proteolytic units Kaken/mg), neuraminidase (I’. cholera, 500.0 units/ml), sphingomyelin, phosphatidylethanolamine, all purine and pyrimidine nucleotides from Calbiochem; chymotrypsinogen (0.19% intrinsic chymotrypsin), trypsin (189.0 units/mg), trypsinogen (1 X crystallized) and soybean trypsin inhibitor from Worthington Biochemicals Corp.; DNAase (2870.0 units/mg), RNAase (100.0 units/mg protein), mercaptoethanol, iodoacetamide, N-ethylmaleimide (NEM), tetranitromethane, 2,4dinitrofluorobenzene, ethylene-glycol-bis(P_aminoethyl ether)-N-N’-tetraacetic acid, dithiothreitol, indomethacin, all fatty acids and steroids from Sigma Chemical Co.; azobenzene sulfenylbromide, p-chloromercuribenzoate @-CMB) and lecithin from Nutritional Biochemicals Corp.; phosphatidylL-serine and lysolecithin from Schwarz-Mann Co.; EHWP Millipore filters (0.5 ,um pore size) from Millipore Corp.; precoated thin-layer silica gel G sheets from Brinkmann Instruments Inc. Purification of / 3H]PGF201 Aliquots of 0.5 PCi of [‘H]PGFza were used in checking the purity by thinlayer chromatography using precoated, 5% boric acid impregnated thin-layer silica gel G sheets and ethyl acetate: acetone: acetic acid (90 : 10 : 1 v/v) solvent system (Anderson, 1969). Following the development of the thin-layer sheets in the above solvent system, the sheets were air dried and scanned with a Packard model 7201 strip scanner. If the purity was less than 95% (judged by the peak area of minor

Properties

of prostaglandin Fzo, receptors

3

peak as compared to the major peak) [3H]PGF2a was repurified by the above described thin-layer chromatography (except 5% boric acid impregnation) prior to use in binding studies. Aliquots of the [3H]PGF2(r stock were diluted to 1 /.&i/ml with redistilled ethanol and stored under nitrogen at -2O’C between uses. Isolation of cell membranes Procedures for the collection of bovine corpora lutea and preparation of the cell membrane fractions were the same as described earlier (Campbell et al., 1972; Rao, 1974a). The composition of the homogenizing buffer is the same as that of the incubation buffer (see binding studies section). The protein content in an aliquot of the membrane fraction was determined by the method of Lowry et al. (1951) using bovine serum albumin as the standard. Binding studies [3H]PGF20, in iedistilled ethanol, was pipetted into 12 X 75 mm disposable glass tubes. The ethanol was blown dry under a stream of nitrogen. Aliquots of membrane fractions containing known amounts of protein were then added to the tubes and incubated (see table and figure legends for additional details). The final composition of incubation buffer (final volume 0.1 or 0.2 ml) was 10 mM TrisHCl, pH 7.0, 250 mM sucrose, 1 mM CaC12, 1 mM dithiothreitol and 0.1% gelatin. Following incubation, 1 ml of 10 mM Tris-HCl buffer, pH 7.0, at 4°C was added to each tube and poured onto Millipore filters positioned on a Millipore manifold under vacuum. The tubes were rinsed twice with l-ml aliquots of 10 mM Tris-HCl buffer and filtered. Finally each filter was washed with 10 ml Tris-HCl buffer at 4°C. The filters were then cut into halves and placed into scintillation vials containing 10 ml of scintillation fluid and counted in a Packard Scintillation counter having a counting efficiency of 22-24%. The scintillation fluid consisted of toluene: Triton X-100: Packard Permafluor (25X) 77 : 20 : 4 (v/v). In each experiment, the nonspecific binding was determined in the presence of excess unlabeled PGFzo, (2.8 X lo-’ M) using the same amounts of membrane protein and [3H]PGF2ry as were used in the total binding tubes. The specific binding, which is presented in all the figures and tables, was obtained by subtracting nonspecific binding from total binding. The nonspecific binding was essentially the same as the binding to the filters in the absence of membrane fractions (blanks) suggesting that nonspecific binding was quite insignificant in these membrane fractions. Thin-layer chromatography of [3H]PGF2, Following incubation of aliquots of 389 pg of membrane protein with 6.2 nM [3H]PGF2a for 1 h at 22”C, the bound and free [3H]PGF2, were separated by centrifugation at 6000g for 15 min. The bound and free [3H]PGF2a! fractions were pooled separately, acidified with 10 ~1 of 2 M citric acid and extracted (3X) with 5 ml of ethyl acetate. The pooled ethyl acetate extracts were dried under a stream of nitrogen and the residue was dissolved in 1 ml of redistilled ethanol. When ex-

4

Ch. V. Rao

tracted bound and free [3H]PGFzor were incubated with aliquots of fresh membranes, the amounts bound were similar to those of unincubated [3H]PGF2a:. When aliquots of extracted bound and free [3H]PGFs, were subjected to thin-layer chromatography, they migrated identically with unincubated [3H] PGFa, on thinlayer chromatographic sheets (impregnated with and without 4% silver nitrate) in two different solvent systems (Green and Samuelsson, 1964; Andersen, 1969). These results suggested that [3H]PGFzol was essentially unchanged during binding phenomenon under the present incubation conditions. Treatment of membranes with enzymes and protein modifying reagents The membrane fractions were preincubated for 1 h at 22°C with various enzymes and protein modifying reagents. Following preincubation, the tubes were centrifuged at 6000g for 15 min. The supernates were aspirated and the pellets were washed with homogenizing buffer (6000g for 15 min). Finally the washed pellets were resuspended in a known volume of homogenizing buffer and aliquots were used for testing the specific binding of [3H]PGF2,. Membranes for controls were incubated without enzymes or protein modifying reagents but subjected to centrifugation similar to treated membranes (see appropriate legends for further details).

RESULTS Dependency of /3H]PGF2, binding on the amount of membrane protein and /3HJPGF2, added The addition of increasing amounts of membrane protein to a fixed amount of [3H]PGF2, resulted in a linear increase in the amount of [3H]PGF2, bound to the membranes. Fig. IA shows that the binding was detectable at concentrations of added [3H] PGFzo, as low as 10-r’ M, increased with increasing concentrations and finally reached saturation at about 2 to 4 X 10e7 M. The Scatchard (1949) plot analysis of [3H]PGF2a binding data presented in fig. 1A revealed that the [3H]PGF2, binding was heterogenous, indicating the presence of high affinity-low capacity and low affinity-high capacity receptors (fig. IB). The apparent dissociation constants (Kd) and the number of available binding sites (N) were calculated from the reciprocal of the slopes and x-axis intercepts, respectively. Free energy changes of +I 1.9 and t10.3 KCal/mol (thermodynamic property of [3H] PGF,,-receptor interaction) were calculated from the apparent equilibrium K, values of high and low affinity sites, respectively, using the equation AF= - RT In K,. Effect of time, temperature and pH on /3H]PGF~, binding At 4°C there was very little f3H]PGFza, binding until 1 h which increased slow-

[3Hl

I

I

I

PGF2a

6

b

I

ADDED

Kd,. N, = Kdp N2 -

C3Hl PGF2a

BOUND,

1.6 x IO-% 117.6 f molrdmg Protrin 2.4 x IO-‘% 750.0 t molrr/mg Protein

M x IO-”

Fig. 1. (A) Dependency of [3H]PGF20r specific binding to bovine corpus luteum cell membranes on the amount of [3H] PGF2, added. Increasing amounts of [3H] PGFza were incubated at 22°C for 2 h with 250 pg membrane protein. Each observation in figs. 1A to 5 represents the mean with its standard error. (B) The Scatchard plot of the binding data presented in fig. 2A.

_J

’ ‘WWQ

50

150 DURATION

OF

INCUBATION,

Min.

Fig. 2. (A) Time- and temperaturedependence of [3H] PGF2, specific binding to bovine corpus luteum cell membranes. Tubes containing 360 pg membrane protein and 3.1 nM 13HjPGF2, were incubated for different times at indicated temperatures. In some tubes, following incubation for 1 h at 22°C excess unlabeled PGFzo was added in 10 ~1 of 0.01 M Tris-HCl, pH 7.0, and incubated again for different lengths of time at 22°C (broken line). (B) Effect of time and temperature on dissociation of [‘H]PGF zo( from bovine corpus luteum cell membrane complex. An aliquot of 43.2 mg of membrane protein in a total volume of 12.0 ml was incubated in a plastic centrifuge tube for 1 h at 22°C with 6.4 nM [3H]PGF2W Following incubation, the tube was centrifuged at 5000g for 15 min and the supernate was aspirated and discarded. The pellet was then resuspended in 120.0 ml of homogenizing buffer. Aliquots of 1.0 ml of the resuspended pellet were pipetted into 12 X 75 mm disposable glass tubes and incubated at indicated temperatures for different times. The amount of 13H] PGFzu bound in 0 time control tubes was 149.6 fmol/mg protein. These tubes were filtered immediately.

Properties

of prostaglandin

I

Fzo, receptors

ly up to 8 h of incubation (fig. 2A). There was a slow but gradual increase in the [aH]PGFaa! binding at 22”C, reaching nearly steady state levels after 1 to 2 h incubation. Further incubation up to 8 h had no effect on steady state levels of binding. At 38”C, however, the initial rate of binding was higher compared to 22°C but binding fell quite rapidly after 20 min of incubation. Since about 90% of the steady state level of binding was obtained following 1 h incubation at 22”, most of the binding studies except equilibrium (for Scatchard analysis) and kinetic binding studies, were conducted for 1 h at 22°C. When washed t3H] PGF2,-membrane complex was incubated in PGF2,-free medium, [3H] PGFzo, dissociated from the membrane complex spontaneously at 38’C, slowly at 22°C and very little at 4°C (fig. 2B). At 38”C, the dissociation of [‘HI PGFzo, from the membrane complex was nearly complete at 1 h of incubation. However, incubation of [3H]PGF2,-membrane complex for 480 min at 22°C and 4°C resulted in dissociation of only 85% and 22% of the membrane-bound [3H] PGF2,, respectively. The addition of excess unlabeled PGF2, following 1 h of incubation of membranes with [3H]PGF2cv resulted in a dissociation of membranebound [3H]PGF20r in a time-dependent manner (fig. 2A). The above results, along with others, suggest that the specific binding of [3H]PGF2(y to its receptors is a really reversible process.

TEMPERATURE,

‘C

Fig. 3. Effect of preincubating bovine corpus luteum cell membranes, at increasing temperatures, on the specific binding of [3H]PGF 2o. Tubes containing 360 pg membrane protein aliquots were preincubated for 15 min at indicated temperatures. Following preincubation, [‘HI PGFzo was added (3.1 nM) and incubated for 1 h at 22°C. The control tubes were not preincubated and the amount bound in these tubes (142.6 fmol/mg protein) was taken as 100%.

8

Ch. V. Rao

Preincubation of membranes for 15 min at increasing temperatures resulted in a gradual but irreversible loss of [3H]PGF2, binding with a complete loss occurring at a temperature of 60°C and above (fig. 3). The temperature-dependent losses of binding during preincubation appear to be due to denaturation of the receptors. Fig. 4 shows that the maximum specific binding occurred at pH 6.4. The specific binding below pH 4.0 and above 9.0 was insignificant. The binding below pH 4.0 was entirely nonspecific. Despite the finding of high specific binding at pH 6.4, routine binding studies were conducted at a medium pH of 7.0. The pH effects on specific and nonspecific binding may have been due to alteration of ionization constants of groups of receptors that are involved in binding interaction and/or pHdependent structural changes in receptor molecules and/or in other membrane components. Rate constants for L3H] PGF,,-membrane interaction As shown earlier, the specific binding of [3H]PGFzol to the membranes

I

I

1

is a time-

1

Fig. 4. Effect of pH of the incubation medium on the specific binding of [ 3H] PGF2, to bovine corpus luteum cell membranes. The membrane fractions containing 250 fig of membrane protein were incubated for 1 h at 22°C with 3.7 nM [3H]PGF2(U. The pH of the incubation medium was maintained by the addition of 0.05 M sodium acetate (3.1 to 7.0) and 0.05 M sodium phosphate (8 to 10.2) buffers.

Properties of prostaglandin Fzol receptors

9

and temperature-dependent process (fig. 2A and B). Since PGF2, receptors are heterogenous with respect to affinity for [3H]PGF2, and it is not possible to resolve association and dissociation which could be ascribed to high and low affmity receptors, a simple assumption was made in the calculation of rate constants: the overall association represents the sum of two separate second order rates and overall dissociation is the sum of two separate first order rates. The rate constants for association, 1 .l X lo4 M-’ s-l, and for dissociation, 3.4 X 10m4 s-l, were calculated from the initial binding velocities (fig. 2A and B), and estimated binding capacities of the membranes (fig. 1B). The apparent K, calculated from rate constants could only give a single value which was closer to the apparent equilibrium K, of low affinity sites. Specificity of / 3H]PGF2, binding As we have shown earlier, the presence of increasing amounts of unlabeled PGFza resulted in a dose-dependent inhibition of [3H] PGF2& binding to the membranes with a complete inhibition occurring at 10e6 M (Rao, -1975a). PGFr, and PGE2 were about equipotent in inhibiting [3H]PGF201 binding to the membranes but these were about 5-fold less effective when compared to unlabeled PGFza. Unlabeled PGEr was much less effective (120-fold) while PGAr and PGB2 were least effective (lO,OOO-fold or less) as compared to unlabeled PGFzc, in inhibiting [3H] PGF2, binding to the membranes (Rao, 1975a,d). hCG, which binds to its own receptors in the same membrane preparations, had virtually no affinity for PGFzo, receptors (Rao, 1975a,d). These results suggest that a 9ol-hydroxy group in the absence of a 5,6double bond primarily determines the affinity for binding to PGF2, receptors (compare PGF2, with PGFr,). The presence of a keto instead of hydroxy group on carbon 9 drastically reduced binding affinity for PGFzo, receptors (see PGEr). However, the presence of a 5,6-double bond in addition to a 9-keto group considerably improved the binding affinity (see PGE2). The absence of an 1 l-hydroxy group and the presence of a 10,l l- or 8,12-double bond results in further reduction in binding affinity (see PGA2, PGBr ). The specificity of t3H] PGFzo, binding is further illustrated in fig. 5. The PGFz~ had very metabolites such as 15-keto-PGF 20! and 13,14-dihydro-15-ketoPGF?, little effect in competing with [3H]PGF 2or for binding to the membranes. 15(S)15Methyl-PGF2,, an analogue of PGF2,, had high affinity but still lower than the parent molecule. The above results in their entirety suggest that functional groups in both the cyclopentane ring and in the side chain play an important role in PGF2,-receptor binding. Various unsaturated fatty acids, indomethacin (PC synthetase inhibitor) and 7-oxa-13-prostynoic acid (PG antagonist) had very little effect in competing with [3H]PGF2, for binding to the membranes. The results suggest that PGF2(U receptors may not be related to PG synthetase activity. Effect of various agents on [3HJPGF201 binding (data not shown) Nucleotides such as CTP and 3’,5’-cyclic AMP (CAMP) had dual effects: at lower

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Properties of prostaglandin Fz,

receptors

11

concentrations they enhanced while at higher concentrations they decreased [‘HI PGFa, binding. The concentration-dependent dual effects were not observed for ATP,UTP or 3’,5’-cyclic GMP (cGMP). A variety of nucleotides (ATP, ADP, AMP, CTP, CDP, CMP, GMP, cCMP, UTP, UDP and UMP) but not others (adenine, cytidine, 3’,5’-cyclic CMP, guanine, GTP, GDP, ITP or uridine) at a concentration of 5 X 10m3 M also inhibited [3H]PGF201 binding to the membranes. The physiological significance of nucleotide effects on [3H] PGF2, binding is unknown at the present time. It should be mentioned at this point, however, that PGF2, has been shown not to increase CAMP production (Marsh, 1970b). We have previously shown that some of these nucleotides at a concentration of 10m3 M enhanced and inhibited [3H]PGE1 and [ 12’1] hCG binding, respectively, to their receptors in these membranes (Rao, 1974a,b). The effect of ionic environment on [3H]PGF2a bindinghas been presented elsewhere (Rao, 1975~). Progesterone, at concentrations which are quite physiological with respect to levels found in corpus luteum (Stormshak and Erb, 1961; Axelson et al., 1975) inhibited [3H] PGFzo, binding to the membranes (Rao, Ch.V., submitted for publication). Although the possible physiological significance of progesterone inhibition of t3H]PGFzol binding can only be speculative at this time, there are prece-

Table 1 Effect of enzyme treatment of bovine corpus luteum cell membranes on the specific binding of [3H]PGF20. Aliquots of 3.1 mg of membrane protein were preincubated with different enzymes (see Experimental procedures). The control and treated membranes (389 pg protein) were then added to tubes containing 6.3 nM [3H]PGF2a: and incubated for 1 h at 22°C. Enzyme: membrane protein ratio was 1 : 3.1 for trypsin, pronase, trypsinogen, chymotrypsinogen, DNAase, lipase, phospholipase C and phospholipase D; 0.78 : 3.1 for RNAase; 50 IU : 3.1 for neuraminidase and 25 IU: 3.1 for phospholipase A. Soybean trypsin inhibitor : trypsin ratio was 2 : 1. There was essentially no loss of binding in the control tubes, therefore taken as 0% inhibition of binding. The amount bound in the control tubes was 13 1.8 fmol/mg protein. Enzyme treatment

Percent inhibition of [3H]PCF2e binding

Trypsin Trypsin + Soybean trypsin inhibitor Pronase Pronase a Trypsinogen Chymotrypsinogen DNAase RNAase Neuraminidase Lipase Phospholipase A a Phospholipase C a Phospholipase D

89.4 +I0.6 3.1 f 1.6 94.8 f 0.3 17.3 * 5.0 20.2 * 0.7 14.3 f 3.4 19.7 + 2.6 22.1 f 1.3 20.5 + 1.9 93.7 f 2.4 94.7 i: 1.9 42.6 f 0.8 1.7 + 0.8

a Boiled at 95°C for 5 mm to remove contaminating

proteolytic

enzymes.

Ch. V. Rao

12

dents set in the literature which show that progesterone can protect the corpus luteurn from luteolytic action of PGFZti (Fuchs and Mok, 1974). Estradiol-170, at approximately the same concentrations as progesterone, had no effect. Effect of enzymes on (3HJPGF2, binding The macromolecular nature of PGFzo, receptors was investigated by preincubating the membranes with various enzymes and subsequently testing for [3H] PGFzo, binding (table I). Proteolytic enzymes such as trypsin and pronase drastically reduced [3H]PGF20, binding. The addition of soybean trypsin inhibitor with trypsin and boiled pronase resulted in much lower losses of [3H]PGFzor binding. Trypsinogen, chymotrypsinogen, DNAase, RNAase and neuraminidase moderately reduced the [3H]PGFzo, binding which may be due to the presence of the active form of the enzymes in the proenzymes and to contaminating proteolytic enzymes in DNAase, RNAase and neuraminidase. The loss of [3H] PGFzo, binding was drastic following treatment of the membranes with lipase and phospholipase A, moderate with phospholipase C and none with phospholipase D. The loss of binding following pretreatment with enzymes and protein reagents (to be discussed later) could be due to specific changes in receptors themselves or secondary to specific changes in other membrane components. The above possibilities were resolved by showing that

Table 2 Restoration of the loss of [ 3H ] PGFzo, specific binding to bovine corpus luteum cell membranes following the treatment of the membranes with phospholipases. Aliquots of 1.9 mg of membrane protein were preincubated with boiled (95°C for 5 min) 12.5 IU of phospholipase A and 0.5 mg of phospholipase C for 1 h at 22°C. Following preincubation, the tubes were centrifuged, supernates aspirated and the pellets were washed once. The washed pellets were resuspended in homogenizing buffer and various phospholipids were added in ethanol (final concentration 9.0%). The tubes were then reincubated at 22°C for 1 h. Following reincubation, the tubes were centrifuged, supernates aspirated and the pellets were washed twice. The washed pellets were then resuspended in homogenizing buffer and aliquots of 389 ng membrane protein were incubated at 22°C for 1 h with 6.4 nM [3H]PGFza. The untreated membranes were processed similar to those of treated membranes except for incubation with enzymes. The amount bound in the control tubes was 66.5 fmol/mg protein. Phospholipid addition (1 mg/ml)

None Lecithin Lysolecithin Phosphatidylserine a Phosphatidylethanolamine Sphingomyelin

Untreated membranes

a

Membranes treated with -

Phospholipase A (70 inhibition of [ 3H]PGFaa. binding)

Phospholipase C

0.0 97.1 94.4 11.6 2.7 0.0

32.7 95.3 82.3 44.7 26.3 5.8

a Added in the form of a suspension.

+ 1.9 + 1.4 ? 2.7 k 0.7

99.0 98.2 100.0 97.2 94.4 97.3

5 0.5 + 0.8 + 0.0 ? 0.4 * 0.9 k 0.6

+ 1.1 * 0.6 f 0.8 * 2.2 + 1.3 i 1.9

Properties

of prostaglandin Fzor receptors

13

loss of binding was attributable to changes in receptor sites alone (Rao, 1976a). Therefore, the above findings suggest that PGF2, receptors are protein in nature and that they require lipids and specific phospholipids for their binding function. Membrane lipids and phospholipids have been reported to participate in binding function of various membrane receptors (Pohl et al., 1971, 1972; Rethy et al., 1971; Barden and Labrie, 1973; Rao, 1974a,b). Therefore, attempts were made to restore the losses of binding in phospholipase A- and C-treated membranes by the addition of specific phospholipids in order to gain some insight into the specific phospholipid requirements for binding. As shown in table 2, the addition of lecithin and lysolecithin, but not others, drastically reduced [3H]PGF2, binding in the untreated membranes. The loss of binding due to phospholipase A treatment of the membranes was not restored by the addition of any of the phospholipids. In phospholipase C-treated membranes, addition of lecithin and lysolecithin further drastically reduced the binding, phosphatidylserine and phosphatidylethanolamine moderately further reduced and restored binding, respectively, while sphingomyelin addition almost completely restored the loss of binding. It was not ascertained however, whether restored binding by sphingomyelin was due to increased affinity or the receptor number or both. The ability of sphingomyelin to restore [3H]PGFzol binding in phospholipase C- but not phospholipase A-treated membranes may be related to the fact that these two phospholipases selectively hydrolyze different parts of the phospholipid molecules (phospholipase A cleaves P-ester linkages whereas phospholipase C cleaves exterior hydrophilic groups (Barden and Labrie, 1973). We have previously reported that sphingomyelin and phosphatidylserine were very effective in restoring [3H]PGE1 binding and [1251] hCG binding, respectively, in phospholipase C-treated membranes (Rao, 1974b,a). These results suggest that PGFza receptors may have specific requirements for sphingomyelin for their binding function. Effect of chemical modification of membrane proteins on [3H]PGF2a binding Membranes were preincubated with several protein modifying reagents and subsequently tested for their effects on [3H]PGF2, binding to determine the possible involvement of protein functional groups in binding interaction (table 3). The binding was significantly reduced following the exposure of membranes to 0.9 mM azobenzene sulfenyl bromide, tetranitromethane, dinitrofluorobenzene and acetic anhydride. The above reagents are known to modify tyrosyl, histidyl, tryptophan residues and amino groups (Means and Feeney, 1971), however, tetranitromethane is highly specific for tyrosyl residues (Sokolovsky et al., 1966). Among several SH group modifying reagents tested, only NEM significantly reduced [3H]PGF201 binding. The decreased binding due to NEM alone may not indicate SH group involvement because this reagent is also known to react with histidyl residues and amino groups (Means and Feeney, 1971). There was a slight reduction in binding following exposure of the membranes to 2.0 M urea. The results with protein modifying reagents in their entirety may sug-

Ch. V. Rao

14

Table 3 Effect of treatment of bovine corpus luteum cell membranes with protein modifying reagents, on the specific binding of [ ‘H]PGF2,. Aliquots of 1.9 mg of membrane protein were preincubated with protein modifying reagents (see Experimental procedures). Aliquots of control and treated membranes (389 ng) were then assayed for binding by incubating for 1 h at 22°C with 6.4 nM [3H]PGFa. The amount of [jH]PGF zol bound in the control tubes was taken as 100%. Reagent

[ 3H]PGFs~ bound (% of control)

Azobenzene sulfenyl bromide a, 0.9 mM Tetranitromethane a, 0.9 mM Dinitrofluorobenzene a, 0.9 mM Acetic anhydride a, 0.9 mM p-Chloromercuribenzoate, 1 mM Iodoacctamide, 10 mM Mercaptoethanol, 10 mM N-Ethylmaleimide, 10 mM Urea, 2.0 M

36.0 14.2 27.5 38.8 107.7 106.1 98.6 35.9 81.3

+ 1.5 * 1.2 * 1.5 f 1.9 * 2.0 f 2.4 c 2.7 t 1.5 + 3.0

a Added in ethanol so that the fina! concentration was 9%. Corresponding controls also contained the same concentration of ethanol. The amount of [3H]PGFa, bound in control tubes was 74.8 fmol/mg protein. Other reagents were added in 0.01 M Tris-HCI with a final pH of 7.0. In the control tubes, the [3H]PGF2(U bound was 125.3 fmol/mg protein.

gest that tyrosyl, histidyl, tryptophan residues and amino groups (any one or all of them), but not SH groups, are involved in the binding interaction of PGFzo, receptors. However, the possible involvement of other residues cannot be completely eliminated.

DISCUSSION During the preparation of this manuscript, a report has appeared in the literature (Powell et al., 1975a) describing the properties of PGFza, receptors in bovine corpora lutea. The following observations: time and temperature effects on binding, dependency of binding on added [3H]PGF2(U and membrane protein, pH of the membrane media and specificity of binding agree with those of Powell et al. (1975a) and Kimball and Lauderdale (1975). However, the major difference between this study and the above two appears to be in the nature of [3H]PGF2, binding. The data presented here, contrary to the above two papers, demonstrate the presence of high affinity-low capacity, low affinity-high capacity PGFza receptors. The possible reasons for this discrepency have been discussed in earlier communications (Rao, 1975a,c). Every batch of membranes prepared in our laboratory in the last twelve months exhibited heterogeneity in [3H]PGFza binding. In different membrane preparations, the Kd for high affinity receptors was in the

Properties of prostaglandin Fza, receptors

15

range of 1 to 5 nM and for low affinity receptors in the range of 16 to 38 nM (Rao, 1975a,c,d). The K,s reported by Powell et al. (1975a) and Kimball and Lauderdale (1975) 5.0 X lo-* M and 2.1 X 10-a M, respectively, correspond to the K, of low affinity receptors (2.4 X lo-* M) found in this study. We have recently demonstrated that observed heterogeneity in [3H] PGFza binding was not due to negative cooperativity or any other phenomena (es., polymerization of ligand, heterogeneity in labeled ligand or lack of true identity between labeled and unlabeled ligand), but that it appears to merely represent the existence of two independent groups of receptor sites with discrete affinities (Rao, 1976b). The outer cell membranes of bovine corpora lutea, besides containing PGFzo receptors, also contain receptors for PGEs (Rao, 1973; Kimball and Lauderdale, 1975) and hCG-LH (Gospodarowicz, 1973; Rao, 1973; Haour and Saxena, 1974; Menon and Kiburz, 1974). While PCs (E, and F,,) had virtually no affinity for hCG-LH receptors and vice versa (Rao, 1974a,b, 1975a,c), there was some overlapping specificity among the PG (PGE and PGFZm) receptors. However, the overlapping specificity (PGEr and PGFza have 120-fold less affinity for each other’s receptors) is such that they could be called ‘discrete’ receptors. While the binding of hCG-LH and PGEs to their membrane receptors in bovine corpora lutea can be correlated with the activation of adenylate cyclase in this tissue (Marsh, 1970a,b), PGFza binding does not appear to be related to this enzyme activity as it has been shown to have no significant effect on the activity of this enzyme (Marsh, 1970b). In vivo, PGs of the F series but not the E series have been shown to be potent in causing luteolysis (demise of corpus luteum) in various subprimate animal species (Weeks, 1972). The physiological role of PGFa, as a luteolysin in sheep (Barcikowski et al., 1974) and guinea pig (Poyser, 1975) has been well documented. It appears quite possible from the specificity and affinity of PGFa,-bovine corpus luteum cell membrane interaction that PGFa,-induced luteolysis involves receptor binding at the cell surface which ultimately leads to the demise of the corpus luteurn. This possibility was further supported by recent findings of Powell et al. (1975b) who demonstrated that the luteolytic potencies of various PCs correlate well with the specificity of PGFza receptor binding. Furthermore, it should also be noted that the apparent Kds of [3H]PGF201 binding (10e9 M and 10e8 M) are in agreement with the circulating utero-ovarian vein levels of PGF (perhaps mostly PGFzo,; 10e9 M and 10-s M) during the end of the bovine estrous cycle and pregnancy (Fairclough et al., 1975; Shemesh and Hansel, 1975) when the luteolysis occurs.

ACKNOWLEDGEMENT The excellent technical assistance of Mr. Fred Carman, Jr., is gratefully acknowledged. This work was supported by grants M74.50 and M75.16 from the Population Council.

16

Ch. V. Rao

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