The human 5-HT1A receptor: comparison of its binding properties in transfected cells and cortical tissue.

1. The binding characteristics of tritium labeled 8-hydroxy-dipropyl-aminotetralin, or [3H]8-OH-DPAT, to the serotonin1A (5-HT1A) receptor in the stably transfected HeLa cell clone HA6 and in human cortical tissue were examined and compared. 2. A series of kinetic studies of [3H]8-OH-DPAT binding to the transfected HA6 cell line demonstrated two components in both the association and the dissociation reactions. 3. In saturation experiments, at least two affinity states were unequivocally detected in the HA6 cell line and the human cortical tissue. Using isotopic dilutions, the binding isotherms were best fitted to a two-site model, and similar affinity values were obtained in both systems (KH approximately 1.1 nM and KL approximately 12-223 nM). 4. Most of the drugs used in competitions inhibited [3H]8-OH-DPAT binding, following a two-site model, and maintained their rank order of binding potency in both systems; that is, 5-HT > or = 8-OH-DPAT > buspirone > pindolol. Inconsistencies, however, were found for the antagonists NAN-190 and pindolol; only one inhibition constant was determined for HA6 cells, but two affinities were detected with cortical tissue. 5. The results indicate that, although data from binding studies using the cell expression system reflect, to a certain extent, those obtained with the cortical tissue, some discrepancies remained. 6. Finally, and in contrast with what is observed with the 5-HT1A receptor expressed in the HA6 cell line, it is possible that different receptors, or subtypes of one receptor, or even uptake sites normally expressed in cortical tissue, could interact with [3H]8-OH-DPAT or the competing drugs or both, thus leading to the observation of additional binding sites.

The human 5-hydroxytryptamine 1A receptor differentially modulates phospholipase C and adenylyl cyclase activities.

In this study, we quantitate and compare the ability of the 5-hydroxytryptamine 1A (5-HT1A) receptor to modulate the activities of phospholipase C and adenylyl cyclase as a function of receptor concentration. We used a single clonal cell line permanently expressing the human 5-HT1A receptor, and progressively depleted the receptor concentration using an alkylating antagonist (N8-bromoacetyl-N1-3'-(4-indolyloxy)-2'-hydroxypropyl-Z-1,8-diamin o-p-methane, (+-) Pindobind). For serotonin-induced phospholipase C stimulation, reductions in receptor number result in dose-response curves that shift downward and rightward, reflecting both a decreasing maximal effect as well as an increasing ED50. In contrast, depletion of more than 95% of the receptors has no effect on the maximal inhibition of forskolin-stimulated adenylyl cyclase activity. Moreover, at all receptor concentrations, the amount of serotonin required to produce half-maximal phospholipase C stimulation is several-fold more than that required to produce half-maximal inhibition of adenylyl cyclase activity. We conclude that the 5-HT1A receptor modulates these two pathways differently, and that the overall response to challenge with serotonin, in terms of both phosphatidyl inositol hydrolysis and cyclic AMP production, is dependent upon receptor number.

Serotonergic antagonists differentially inhibit spontaneous activity and decrease ligand binding capacity of the rat 5-hydroxytryptamine type 2C receptor in Sf9 cells.

The activities of serotonergic antagonists as inverse agonists at the rat 5-hydroxytryptamine (5-HT)2C serotonin receptor were compared with their potencies in promoting receptor "down-regulation," after expression of the recombinant receptor in the baculovirus/Sf9 insect cell system. Baculovirus expression yielded high levels of 5-HT2C receptors (up to 10(6) receptors/cell), which were functionally coupled to polyphosphoinositide turnover in Sf9 cells through a pertussis toxin-insensitive pathway. The expressed receptor exhibited spontaneous activation of inositol phosphate production, which was inhibited in a dose-dependent manner by serotonergic antagonists, consistent with inverse agonist activity. The potencies of antagonists as inverse agonists correlated with their respective binding affinities determined in competition binding studies with membrane preparations. The maximal inhibition of spontaneous activity ranged from 32% inhibition for mianserin to no effect for spiroxatrine, indicating that antagonists differ in their intrinsic inverse efficacies. Antagonist treatment of intact Sf9 cells or membranes containing the 5-HT2C receptor, followed by washout of residual drug, resulted in a decrease (up to 90%) in the number of binding sites for [3H]mesulergine and [3H]5-HT, with no change in the affinity for [3H]mesulergine. The decrease in binding was irreversible, was not due to the presence of residual antagonist, and was not observed after treatment with agonists. This effect of antagonists in membranes was dose dependent, but the rank order of potency was clearly different from that for inverse agonist activity, indicating that the two effects reflect distinct actions of antagonists at the 5-HT2C receptor. The relative abilities of antagonists to produce loss of binding showed a good correlation with their reported abilities to down-regulate 5-HT2 receptors in vivo after chronic treatment, suggesting that these actions reflect the same underlying process.

Serotonin upregulates mitogen-stimulated B lymphocyte proliferation through 5-HT1A receptors.

Serotonin is a well-known neurotransmitter and neuroimmunomodulator which has been reported to modulate T cell and NK cell proliferation. In this study we investigated whether serotonin could regulate mitogen-stimulated proliferation of the mature B lymphocyte. Mouse and rat spleen cells were cultured with serotonin in the presence or absence of a combination of Escherichia coli lipopolysaccharide and dextran sulfate, and proliferation was assessed by [3H]thymidine uptake or propidium iodide staining of DNA. Serotonin alone had no effect on spleen cell proliferation, while it increased mitogen-stimulated B cell proliferation in a dose- and time-dependent manner. These effects were reproduced by the selective 5-HT1A receptor agonist 8-OH-DPAT. Serotonin- or 8-OH-DPAT-induced increase in proliferation could be blocked by the 5-HT1A receptor antagonists (+)WAY 100135 and propranolol. Moreover, lipopolysaccharide-activated mouse spleen cells expressed specific binding sites for [3H]8-OH-DPAT. These results show that serotonin upregulates mitogen-stimulated B lymphocyte proliferation through 5-HT1A receptors, thus providing an important link between this neurotransmitter and the immune system.

Agonist/antagonist interactions with cloned human 5-HT1A receptors: variations in intrinsic activity studied in transfected HeLa cells.

The characteristics of 5-HT1A-recognition sites and receptor-mediated release of intracellular calcium were established in two transfected HeLa cell lines (HA 6 and HA 7) expressing different levels of human 5-HT1A receptors (about 3000 and 500 fmol/mg protein, Fargin et al. 1989; 1991; Raymond et al. 1989). The pharmacological profiles of the binding (determined with [3H]8-OH-DPAT) and the calcium response (measured using Fura-2) were clearly of the 5-HT1A type. Compounds such as 5-HT, 5-CT and 8-OH-DPAT acted as full agonists on the calcium response in both HeLa cell lines. In addition, methiothepin, pindolol, NAN 190 and SDZ 216-525 (Seiler et al. 1991) acted as silent and potent antagonists. Marked differences were observed in the responses mediated in the two cell lines. EC50 values of agonists (particularly 5-HT, 5-CT, flesinoxan and 8-OH-DPAT) were higher in HA 7 cells (up to 80-fold) than in other 5-HT1A receptor models (e.g. inhibition of adenylate cyclase in calf hippocampus). Further, a variety of compounds (ipsapirone, buspirone, spiroxatrine, MDL 73005) acted as agonists in HA 6 cells, whereas they behaved as silent antagonists in HA 7 cells (which express fewer receptors). By contrast, KB values for antagonists were comparable in HA 6 and HA 7 cells. The present data show that EC50 values and intrinsic activity for a given drug are subject to large variations depending on the number of receptors expressed in the target tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Dual coupling of the cloned 5-HT1A receptor to both adenylyl cyclase and phospholipase C is mediated via the same Gi protein.

The cloned 5-HT1A receptor, stably expressed in HeLa cells, has been shown to mediate the effects of 5-hydroxytryptamine (5-HT) to inhibit cAMP formation and to stimulate the hydrolysis of phosphatidylinositol. Both responses were found to be pertussis toxin sensitive. We have examined these two responses in membranes derived from these cells and show that the 5-HT1A receptor can directly regulate the activity of adenylyl cyclase and phospholipase C in response to agonist. In order to examine whether the same or distinct guanine nucleotide-binding regulatory protein(s) (G protein) are involved in these two signal transduction pathways, we used anti-peptide antibodies recognizing the alpha-subunits of Gi1, Gi2, Gi3 as specific tools, since these pertussis toxin substrates are expressed in HeLa cells. These antibodies have previously been shown to prevent receptor-G protein coupling by binding to the regions of G proteins which are putatively involved in interaction with receptors. Our results indicate that the Gi proteins, but preferentially Gi3, mediate the effects of 5-HT both to inhibit adenylyl cyclase and to stimulate phospholipase C. These findings demonstrate that the same receptor interacting with the same G protein can regulate several distinct effector molecules.

The 5-HT1A receptor: an overview of recent advances.

Progress in the field of neuronal receptor research has accelerated during the last few years due to developments in pharmacology and molecular biology. This is particularly true in the case of the serotonin 5-HT1A receptor. In 1983 the very selective, high affinity 5-HT1A agonist 8-OH-DPAT was developed which allowed the pharmacology and distribution of the 5-HT1A receptor in the central nervous system of the rat and man to be extensively characterized. By 1987, the gene encoding this receptor protein was cloned and sequenced, allowing not only elucidation of its structure, but also better insight into the nature of its coupling to transmembrane signal transduction systems. Thus in a short period of time considerable knowledge has accumulated on how serotonin exerts its functions in the central nervous system via the 5-HT1A receptor. In the present review we will briefly discuss some of the latest developments regarding the 5-HT1A receptor.

A potent and selective photoaffinity probe for the anti-estrogen binding site of rat liver.

The anti-estrogen binding site (ABS) is an apparently ubiquitous component of cells that has been shown to be intimately linked with the antiproliferative effects of certain antiestrogenic compounds, like tamoxifen, which is currently used for the treatment of breast cancer. However, the identification and in vitro study of this novel protein has been hampered to date by a lack of convenient probes that will efficiently label the molecule in nonpurified preparations. Thus, using a selective ABS ligand (4-benzylphenoxy-N-ethylmorpholine, MBPE) as starting material, we synthesized a photosensitive azido derivative, [(2-azido-4-benzyl)phenoxy]N-ethylmorpholine (azido-MBPE) that can be prepared in a tritiated form. Azido-MBPE has a high affinity for ABS (Kd = 3 nM), identical to that of tamoxifen, and covalently labels 5 and 12% of membrane-bound and detergent-solubilized ABS, respectively. Its incorporation is selectively and competitively inhibited by other ABS ligands (tamoxifen greater than nitromifen greater than hydroxytamoxifen). [3H]Azido-MBPE potently photolabels either membrane-bound or detergent-solubilized ABS as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under denaturing conditions revealing specific photoincorporation in a protein band of Mr = 40,000. This molecular weight is approximately two times lower than what we observed previously for ABS preparations studied under nondenaturing conditions and postlabeled with [3H]tamoxifen (Mr = 80,000-110,000). In chromatofocusing experiments with photolabeled ABS, a single specifically labeled protein fraction migrating with a pI of 6.4 was found to exhibit a Mr of 40,000 when subsequently electrophoresed on sodium dodecyl sulfate-polyacrylamide gels. These results indicate that [3H]azido-MBPE is a specific high affinity probe of ABS that will prove useful in the ultimate identification of this protein.

The anti-proliferative properties of 4-benzylphenoxy ethanamine derivatives are mediated by the anti-estrogen binding site (ABS), whereas the anti-estrogenic effects of trifluopromazine are not.

We compared the anti-proliferative properties of 4-benzylphenoxy-N ethyl morpholine (morpho-BPE) and trifluopromazine (TFP) on both the human breast cancer cell lines, MCF7, and its tamoxifen-resistant variant RTx6. We found that the calmodulin antagonist trifluopromazine (TFP) which bound ABS weakly, inhibited MCF7 cell growth but did not follow the relationship observed for diphenylmethane derivatives between MCF7-inhibitory potencies and their Ki. Regarding the tamoxifen-resistant RTx6 cells, TFP but not morpho-BPE induced inhibition of the proliferation. Using a tritiated derivative of morpho-BPE, two distinct binding sites could be demonstrated. Indeed, a low affinity binding site was present in both cell lines whereas a high affinity binding site was mainly found in MCF7 cells although being in lower concentration (less than 10%) in RTx6 cells. Both tamoxifen and TFP displaced morpho-BPE from the two binding sites. The uptake and efflux of the tritiated drug were similar in the two cell lines. The drug did not appear to be metabolized. We concluded that TFP and morpho-BPE belong to distinct classes of molecules and that ABS mediates the anti-proliferative action of diphenylmethane derivatives but not the inhibitory effect of the calmodulin antagonist TFP.

The human 5-HT1A receptor expressed in HeLa cells stimulates sodium-dependent phosphate uptake via protein kinase C.

Regulation of phosphate uptake was studied in HeLa cell lines after transfection with DNA encoding the human 5-HT1A receptor. Phosphate uptake was saturable and greater than 90% sodium-dependent, with Vmax approximately 30-35% without changing Km. Treatment with 5-HT or the 5-HT1A-specific agonist 8-OH-2-(di-n-propylamino)1,2,3,4-tetrahydronaphthalene increased Vmax approximately 40% without affecting Km. This effect was blocked by pretreatment with the 5-HT1 antagonists, methiothepine and spiperone, or pertussis toxin. Surprisingly, the stimulation was not secondary to an inhibition of adenylyl cyclase because 5-HT stimulated phosphate uptake approximately 20% in the presence of 1 mM 8-Br-cAMP. Rather, the primary pathway linked to the stimulation of phosphate uptake involved activation of protein kinase C because (i) 5-HT measurably activated protein kinase C in these cells, (ii) activators of protein kinase C (phorbol esters and diacylglycerol analogues) stimulated phosphate uptake in these cells (iii) the half-maximal doses for 5-HT-induced phosphatidylinositol hydrolysis and stimulation of phosphate uptake were virtually equivalent, and both effects were equally sensitive to pertussis toxin, and (iv) the stimulation was markedly attenuated in cells made deficient in protein kinase C. These results demonstrate that the stimulation of phosphatidylinositol hydrolysis by the 5-HT1A receptor can generate physiologically measurable effects on cellular transport and suggest that such accessory pathways may play a prominent role in signal transduction.

Effector coupling mechanisms of the cloned 5-HT1A receptor.

The signal transduction pathways of the cloned human 5-HT1A receptor have been examined in two mammalian cell lines transiently (COS-7) or permanently (HeLa) expressing this receptor gene. In both systems, 5-hydroxytryptamine (5-HT, serotonin) mediated a marked inhibition of beta 2-adrenergic agonist-stimulated (80% inhibition in COS-7 cells) or forskolin-stimulated cAMP formation (up to 90% inhibition in HeLa cells). This serotonin effect (EC50 = 20 nM) could be competitively antagonized by metitepine and spiperone (Ki = 81 and 31 nM, respectively) and could also be blocked by pretreatment of cells with pertussis toxin. In both cell types, 5-HT failed to stimulate adenylyl cyclase through the expressed receptors. In HeLa cells, 5-HT also stimulated phospholipase C (approximately 40-75% stimulation of formation of inositol phosphates). Again, this effect was inhibited by metitepine. However, the EC50 of 5-HT was considerably higher (approximately 3.2 microM) than that found for inhibition of adenylyl cyclase. Both pathways were demonstrated to be similarly affected by pertussis toxin. These findings indicate that like the M2 and M3 muscarinic cholinergic receptors, the 5-HT1A receptor can couple to multiple transduction pathways with varying efficiencies via pertussis toxin-sensitive G-proteins. The lack of stimulation of cAMP formation by this 5-HT1A receptor may suggest the existence of another pharmacologically closely related receptor.

Identification of the ligand-binding subunit of the human 5-hydroxytryptamine1A receptor with N-(p-azido-m-[125I] iodophenethyl)spiperone, a high affinity radioiodinated photoaffinity probe.

The ligand-binding subunit of the human 5-hydroxytryptamine1A (5-HT1A) receptor transiently expressed in COS-7 cells and of the native human 5-HT1A receptor derived from hippocampus and frontal cortex were identified by photoaffinity labeling with N-(p-azido-m-[125I]iodophenethyl)spiperone [( 125I]N3-NAPS), previously characterized as a high affinity radioiodinated D2-dopamine receptor probe. The identity of the ligand-binding subunit was confirmed by immunoprecipitation with an antipeptide rabbit antiserum, JWR21, raised against a synthetic peptide derived from the predicted amino acid sequence of the putative third intracellular loop of the human 5-HT1A receptor. In transiently transfected COS-7 cells expressing 14 +/- 3 pmol/mg of protein human 5-HT1A receptors, a single broad 75-kDa band was photoaffinity labeled by [125I]N3-NAPS. This band displayed the expected pharmacology of the 5-HT1A receptor, as evidenced by the ability of a series of competing ligands to block [125I]N3-NAPS photoincorporation. Moreover, antiserum JWR21 specifically and quantitatively immunoprecipitated the 75-kDa photoaffinity-labeled band from a soluble extract of the transfected COS-7 cell membranes, further confirming its identity. Finally, utilizing a combination of photoaffinity labeling and immunoprecipitation, the native ligand-binding subunit of 62-64 kDa was identified in human hippocampus and frontal cortex. The availability of the high specific activity, high affinity, photoaffinity ligand [125I]N3-NAPS and of a potent immunoprecipitating antiserum (JWR21) should greatly facilitate the biochemical characterization of the human 5-HT1A receptor.

Solubilization of a tamoxifen-binding protein. Assessment of its molecular mass.

Recent findings point to a role of Antioestrogen-Binding Site (ABS) in some of the growth-modulatory effects of antioestrogens. In the present study, a method for the solubilization of ABS from rat uterus microsomal fractions by using 3-(3-cholamidopropyl)dimethylammonio-1-propanesulphonate (CHAPS; 20 mM) and KCl (0.4 M) is described. Decreasing the CHAPS concentration below the critical micelle concentration led to long-term stabilization of the protein. All of the membrane-bound ABS was recovered in the extract, and only one class of binding site, with a high affinity for [3H]tamoxifen (KA = 5 x 10(8) M-1) was detectable. This binding was time-dependent and reversible: at 4 degrees C, the association rate constant was ka = 7.2 x 10(4) M-1.s-1, and the reverse rate constant was kd = 1.0 x 10(-4) s-1. Solubilized ABS exhibited an affinity and specificity similar to those of the membrane-bound sites. Under disaggregating conditions, solubilized ABS had an apparent sedimentation coefficient, s20,w, of 5.2 S and a Stokes radius of 6.4 nm. From these two values, molecular masses of 160,000 Da for the detergent-ABS complex, and 110,000 for the protein moiety, were estimated. Assessment of the size of the membrane-bound ABS by a radiation inactivation technique is also described. The 'radiation inactivation size', corresponding to the mass of 1 mol of protein structure(s) whose associated tamoxifen-binding activity is abolished after a single hit by ionizing radiation, was estimated to be 80,000 Da.

The genomic clone G-21 which resembles a beta-adrenergic receptor sequence encodes the 5-HT1A receptor.

The recent cloning of the complementary DNAs and/or genes for several receptors linked to guanine nucleotide regulatory proteins including the adrenergic receptors (alpha 1, alpha 2A, alpha 2B, beta 1, beta 2), several subtypes of the muscarinic cholinergic receptors, and the visual 'receptor' rhodopsin has revealed considerable similarity in the primary structure of these proteins. In addition, all of these proteins contain seven putative transmembrane alpha-helices. We have previously described a genomic clone, G-21, isolated by cross-hybridization at reduced stringency with a full length beta 2-adrenergic receptor probe. This clone contains an intronless gene which, because of its striking sequence resemblance to the adrenergic receptors, is presumed to encode a G-protein-coupled receptor. Previous attempts to identify this putative receptor by expression studies have failed. We now report that the protein product of the genomic clone, G21, transiently expressed in monkey kidney cells has all the typical ligand-binding characteristics of the 5-hydroxytryptamine (5-HT1A) receptor.

Further evidence for a biological role of anti-estrogen-binding sites in mediating the growth inhibitory action of diphenylmethane derivatives.

Several diphenylmethane derivatives have been synthesized with variable affinities for Anti-estrogen Binding Sites (ABS) but not for the estrogen receptor. Using these molecules as probes it is shown that their binding affinities for ABS correlate with their abilities to inhibit the growth of MCF-7 human breast cancer cells. In contrast they have no influence on the proliferation of tamoxifen-resistant variant cells (RTx6) in which ABS are undetectable. These data support the conclusion that ABS has a functional role in the anti-proliferative effect of triphenylethylene anti-estrogens and structurally related compounds.

Antiestrogens, different sites of action than the estrogen receptor?

Nonsteroidal antiestrogens display antagonistic as well as agonistic properties when compared with estrogens. These observations and studies of their molecular interactions suggest that part of their activity is mediated through estrogen receptor (ER)-mediated pathways. However, other data, concerning mainly effects on growth and cellular proliferation, cannot be explained on this basis. Recent investigations have shown binding of these molecules to a number of other intracellular binding sites beside ER. Using different cell variants of the human breast cancer cell line MCF-7 we could confirm the peculiar role of one of these sites, the specific antiestrogen-binding site (ABS). Published data on ABS are critically reviewed.

Different interaction of estradiol and antiestrogens with the estrogen receptor of rat uterus.

Cytosoluble estradiol-receptor (ER) complexes obtained from uteri of castrated rats show a 4.3 S sedimentation coefficient when analysed by sucrose gradient in high salt buffer both in the absence and presence of 0.1 M ammonium thiocyanate. After incubation with nuclei, the complexes sediment at 4.3 S and at 3.2 S, in the absence and presence of 0.1 M ammonium thiocyanate respectively. This structural modification of ER is also confirmed by gel filtration analysis. In similar conditions hydroxytamoxifen-ER complexes apparently do not undergo such a modification. However, increasing the molarity of the chaotropic ion up to 0.5 M shows that, in fact, the modification occurs. Sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis of covalently linked tamoxifen aziridine-ER 'complexes' confirms that the nuclear treatment reduces the apparent molecular weight of ER from 62 000 to 47 000. These data demonstrate that, in vitro, nuclear ER is cleaved into a smaller molecular form and that the receptor fragments can be held together by the triphenylethylene antiestrogen hydroxytamoxifen.

Dissimilarities between the uterine estrogen receptor in cytosol of castrated and estradiol-treated rats.

The estrogen receptor (ER) in its native state appears oligomeric and can be dissociated by salt into a monomer with a mol wt of 50,000-80,000. Lower molecular weight fragments have also been observed but are considered to result from ER proteolysis by tissue proteases. Three-month-old Sprague-Dawley rats have been studied after castration, estradiol treatment (3 days at 25 micrograms/day), or during the normal estrus cycle. The uterine cytosol was labeled with [3H]estradiol, and the [3H]estradiol-receptor complexes were studied by sucrose gradient centrifugation, gel filtration, diethylaminoethyl (DEAE)-Trisacryl and phosphocellulose chromatography, and kinetic experiments. In low salt buffer in presence of molybdate (20 mM), uterine ER of castrated and treated rats could not be differentiated by sucrose gradient centrifugation (9.9S), gel filtration (7.7 nm), or DEAE-Trisacryl and phosphocellulose chromatography, but the dissociation rate at 23 C was lower for castrated than for treated rats (t1/2 = 45 and 27 min, respectively). In high salt buffer (0.4 M KCl) in presence of molybdate (20 mM), no difference was apparent by sucrose gradient centrifugation (3.7S) or gel filtration (3.4 nm), but again the dissociation rate at 23 C was lower for castrated than for treated rats (t1/2 = 102 and 45 min, respectively). In high salt buffer in presence of molybdate and ammonium thiocyanate (0.5 M), differences were observed by sucrose gradient centrifugation (3.6 and 3.2S), gel filtration (3.2 and 2.6 nm), and dissociation rate assays at 10 C (t1/2 = 24 and 12 min) for castrated and treated rats. The calculated wts were 50,000 and 35,000, respectively. Protection from limited proteolysis by molybdate, contained in all buffers used, plus various protease inhibitors, particularly leupeptin, as well as mixing tissues before homogenization, suggested that the 35,000-dalton monomer was not a product of protease action formed after cell breakage. ERs covalently labeled with [3H]tamoxifen aziridine also showed different entities of 62,000 and 47,000 mol wts, respectively, on denaturing polyacrylamide gels. In vitro activation and transformation were demonstrated for the two types of [3H]estradiol-receptor complexes by sucrose gradient centrifugation analysis and affinity for phosphocellulose and purified nuclei. Finally, it has been shown that both entities coexisted in different proportions during the diestrous (1:2) and proestrous (1:1) phases of the estrus cycle; only the low mol wt component was present during the estrous phase.