Monoclonal antibodies against rabbit mammary prolactin receptors. Specific antibodies to the hormone binding domain

Three monoclonal antibodies (M110, A82, and A917) were obtained by fusing myeloma cells and spleen cells from mice immunized with partially purified rabbit mammary gland prolactin (PRL) receptors. All 3 antibodies were capable of complete inhibition of 125I-ovine prolactin (oPRL) binding to rabbit mammary PRL receptors in either particulate or soluble form. M110 showed slightly greater potency than oPRL in competing for 125I-oPRL binding. These antibodies also inhibited PRL binding to microsomal fractions from rabbit liver, kidney, adrenal, ovary, and pig mammary gland, although A82 showed poor inhibition in pig mammary gland. There was no cross-reaction of any of the 3 monoclonal antibodies (mAbs) for the other species tested: human (T-47D breast cancer cells) and rat (liver, ovary). In order to confirm that these antibodies are specific to the binding domain, antibodies were purified, iodinated, and binding characteristics were investigated. 125I-M110 and 125I-A82 binding was completely inhibited by lactogenic hormones, whereas nonlactogenic hormones did not cross-react. Competition of 125I-M110 by oPRL (ID50 = 0.44 nM) was comparable to that of 125I-oPRL by unlabeled oPRL (ID50 = 0.35 nM), while 125I-A917 binding was only partially competed (30-60%) by lactogenic hormones. Tissue and species specificity of labeled antibody binding paralleled results of binding inhibition experiments using 125I-oPRL. In addition, A82 and A917 completely inhibited 125I-M110 binding. In contrast, 125I-A82 binding was stimulated by A917 and 125I-A917 binding was stimulated by A82. These findings indicate that monoclonal antibodies can be readily prepared from partially purified PRL receptors from rabbit mammary gland; two antibodies (M110 and A82) are hormone binding site specific while the other (A917) binds a domain partially but not entirely distinct from the hormone binding site, and that all three antibodies have strong species specificity.     

Two monoclonal antibodies against prolactin-receptor are internalized in epithelial mammary cells without mimetic prolactin effect on casein secretion*

Summary— Prolactin exerts an early stimulatory effect on casein secretion which was qualified as a secretagogue effect. After binding to its receptor, the hormone transits intracellularly through the mammary epithelial cell. When this transit is slowed down the secretagogue effect does not occur. Different monoclonal antibodies which bind to the rabbit prolactin receptor have been previously developed. One of them (A917) mimics prolactin effect on casein gene expression. Another (M110) blocks this prolactin effect. In order to study the respective role of the hormone and its receptor, we have examined the binding of the two monoclonal antibodies (M110 and A917), labeled with biotin or colloidal gold, to the receptor of lactating rabbit mammary epithelial cells in incubation. Subsequently, the intracellular movement of these antibodies and the secretory response have been measured. Irrespective of the labeling (biotin or colloidal gold) or the preparation of tissues (fragments or enzymatically dissociated cells), Ml 10 and A917 bound to the basal membrane of mammary epithelial cells. However, only M110 bound to apical membrane of dissociated cell when this membrane was in direct contact with the incubation medium, showing that the two antibodies discriminate the receptor located on the apical membrane. Following internalization, each antibody was carried via a peculiar pathway. M110 remained associated with the cells during a 1-h incubation, mainly in endosomes, multivesicular bodies and lysosomes like vesicles. In contrast, A917 was very quickly detectable in endosomes, multivesicular bodies and vesicles of the Golgi region and was carried throughout the cell to the lumen of the acini. M110 and A917 were extremely rare in secretory vesicles containing casein micelles. When mammary fragments pulse labeled for 3 mm with ³H-leucine were chased for 60 mm in the presence of prolactin, M110 or A917, only prolactin was able to increase casein secretion. These results show that two monoclonal antibodies against prolactin receptor are internalized after binding to the surface of the mammary cell. They are carried intracellularly by different routes. Internalization of these antibodies is not sufficient to mimic the secretagogue effect of prolactin.     

In vivo lactogenic effects of anti prolactin receptor antibodies in pseudopregnant rabbits

Antibodies generated against partially purified prolactin receptors from rabbit mammary gland membranes were tested for their effects on prolactin binding to receptors and for their in vivo biological potencies. These antibodies are able to inhibit prolactin binding to crude rabbit mammary gland membranes. When administered intravenously or intramuscularly to pseudopregnant rabbits, they induce respectively an accumulation of beta-casein or an enhancement of beta-casein synthesis and mRNA concentration in the mammary gland. Moreover the stimulatory effect of these anti-prolactin receptor antibodies on casein synthesis is totally abolished by a simultaneous treatment with progesterone, which is a potent in vivo inhibitor of prolactin action. These results better establish the prolactin-like activities of these antibodies previously observed in vitro and give strong support to the hypothesis that prolactin molecule is not required beyond the initial binding to its receptor to induce hormonal effects.     

Prolactin receptor: identification of the binding unit by affinity labelling and characterization of poly- and monoclonal antibodies

The prolactin receptor localized in rabbit mammary gland membranes has been identified by affinity labelling using covalent cross-linking agents such as a unique protein chain of approximately 32,000 daltons. After partial purification (5,000-fold) of these receptors from mammary gland homogenate, polyclonal antibodies, which specifically and completely inhibit prolactin binding in all organs and in all species studied, were raised. These antibodies possessed prolactin-like biological activity (casein synthesis) on rabbit mammary gland explants. Monoclonal antibodies specifically directed against the binding domain of the receptor were also obtained. These antibodies were more species-specific than the polyclonal antibodies. The most potent (M110) possessed higher affinity than prolactin for the receptor and could be a very effective tool to elucidate the structure of the receptor and its immunological detection.     

Antiprogesterone and antiglucocorticoid actions of RU 486 on rabbit mammary gland explant cultures. Evidence for a persistent inhibitory action of residual progesterone upon the mammary tissue

The antiprogesterone and antiglucocorticoid compound RU 486 added to pregnant rabbit mammary gland explant cultures had no effect alone but significantly stimulated casein production in the presence of ovine prolactin (PRL) in a dose dependent manner. This stimulation was inhibited by progesterone (Pg) and the Pg agonist R5020. When the explants were cultured for 5 days with two changes of medium, to eliminate all steroids, and hormones added afterwards, the effect of PRL was potentiated, Pg was no longer inhibitory and RU 486 had no effect, RU 486 also could inhibit the stimulatory action of glucocorticoids added to the cultures along with PRL. The compound was able to displace [3H]dexamethasone and [3H]R 5020 from mammary gland glucocorticoid and Pg receptors respectively and proved to have a high relative binding affinity (RBA) for both receptors when compared with typical ligands for each receptor. The RBAs of RU 486 and the steroids used in this study to mammary gland glucocorticoid and Pg receptors correlated well with the ability of RU 486 to block their biological activities. These results demonstrate that RU 486 has both antiglucocorticoid and antiprogesterone activities in pregnant rabbit mammary glands as well as the existence of a strong inhibitory residual action of Pg in the gland that persists during the first 48 h of culture and that can be eliminated by RU 486 or after several days of culture with no hormones.     

Immunological recognition of the prolactin receptor: identification of a single binding unit of molecular weight approximately 42,000

Different polyclonal and monoclonal antibodies against the rabbit mammary prolactin (PRL) receptor were previously obtained that totally inhibited PRL binding in the rabbit mammary gland. Only polyclonal antibodies were shown to immunoprecipitate preformed PRL--receptor complexes in solubilized mammary membranes suggesting that they also recognized domains outside of the PRL binding site of the receptor. When partially purified PRL receptor preparations from both rabbit and pig mammary tissues were iodinated, immunoprecipitated and subsequently analyzed by SDS--PAGE, a single component of molecular weight approximately 42,000 was specifically recognized by all the anti-PRL receptor antibodies. This unit was the only component immunoprecipitated by the monoclonal antibody M 110. Its identification was not impaired by using reducing or non-reducing conditions. Moreover, a further purification of the [125I]-labeled receptor preparations from both species by a second PRL affinity chromatography selected a single binding unit of the same molecular weight. In contrast, polyclonal antibodies immunoprecipitated additional components apart from the 42,000 unit, especially one unit of molecular weight 70,000-80,000 in both species. We conclude that rabbit and pig mammary PRL receptors exhibit striking immunological similarities. Both contain a single binding unit of molecular weight approximately 42,000 that is not linked to other units via disulfide bridges. This binding unit could be associated with a larger component of MW 70,000-80,000 in the holo receptor.     

Water-soluble prolactin receptors from porcine mammary gland

Two types of prolactin receptors were identified in sow mammary gland. When light membranes were prepared on a discontinuous sucrose gradient (0.3 and 1.7 M) and then diluted and washed with 0.3 M sucrose solution, a large amount (about 50%) of receptors were released from membranes and appeared in the supernatant fraction. These two forms (hydrophobic and water-soluble) of receptors were characterized as having the same binding specificity for lactogenic hormones and a similar affinity constant for ovine prolactin (K alpha approximately 10-12 X 10(9) M-1). Polyclonal antibodies and one monoclonal (mAb M110) antibody, obtained against partially purified prolactin receptors from rabbit mammary gland, cross-reacted effectively with sow mammary receptors. They completely inhibited the specific binding of [125I]oPRL to membrane and water-soluble receptors. The present studies indicate that the two types of sow prolactin receptors could represent the same molecular entity and confirm that prolactin receptors from rabbit and sow mammary gland exhibit numerous antigenic similarities.     

Biological actions of monoclonal luteinizing hormone/human chorionic gonadotropin receptor antibodies.

Several recent studies have elucidated the structure of the mammalian LH/hCG receptor; as reported in the present work, we have developed a series of monoclonal antibodies (mAbs) against the rat ovarian LH/hCG receptor using highly purified receptor as immunogen and by screening hybridomas with purified LH/hCG receptors. The mAbs were able to specifically immunoprecipitate LH/hCG receptors from solubilized preparations of rat ovarian membranes as well as from partially purified preparations. Western blotting with mAb P1B4 detected a probable receptor dimer and a receptor fragment in rat and porcine ovarian tissue but not in other tissues. This mAb also partially inhibited hCG binding to rat and porcine ovarian tissues. The receptor mAbs were able to inhibit hCG-induced progesterone synthesis in cultured human and porcine granulosa cells without affecting cAMP- and FSH-induced progesterone synthesis. The mAb P1B4 was used to demonstrate that the majority of ovarian receptors are internalized after hCG treatment and that in pseudopregnant rats receptors are present in the rough endoplasmic reticulum and in microvesicles. Bovine corpus luteal cells also contained P1B4 binding sites, as detected by immunohistochemical technique. Taken together, these results suggest that the mAbs are specific for the LH/hCG receptor, mAb P1B4 recognizes an epitope that is highly conserved among mammals, and this epitope is probably in the extracellular domain.     

Temperature dependence of prolactin endocytosis and casein exocytosis in epithelial mammary cells

As previously reported in epithelial mammary cells of lactating rabbit, prolactin exerts a stimulatory effect on casein secretion. After binding to a membrane receptor, the complex hormone-receptor is internalized in mammary cells. Peptide hormone action involves the generation of second messengers. These second messengers can be emitted as soon as hormone is linked to the membrane receptor. However, it is not excluded that endocytosis and transfer of prolactin inside the cell take part in the emission of second messenger and related secretory response. In order to precise intracellular transport pathways in the lactating mammary cell, we have examined the effects of reduced temperature on the one hand on prolactin endocytosis, on the other hand on casein secretion and on the stimulating effect of prolactin on casein secretion. Endocytosed prolactin was cytochemically localized mainly on the plasma membrane at 4 degrees C. At 25 degrees C, the hormone accumulated, during 60 min, in endosomes and multivesicular bodies. At 37 degrees C, prolactin was detectable after 15 and 30 min inside the cells and disappeared after 60 min. Transport and exocytosis of secretory proteins were only partly inhibited at 25 degrees C as attested by autoradiography localization and biochemical assays of newly synthesized caseins. However, at 25 degrees C, prolactin was no more able to stimulate casein exocytosis. These results show that intracellular transport of prolactin and secretagogue effect of the hormone does not proceed at 25 degrees C. However, secretory mechanisms of the cell are always able to be stimulated by exogenous arachidonic acid at this temperature. Low temperature appears as a good means to study intracellular transport in the mammary cell.     

Partial Proteolytic Digestion of the Mammary Prolactin Receptor: Identification of Smaller Prolactin Binding Fragments

Abstract

Partial proteolytic digestion of the mammary prolactin (PRL) receptor was used to generate receptor fragments and analyze their immunoreactivity and PRL binding properties. Tryptic digestion of the PRL receptor produced two immunoreactive fragments (Mr ≈ 30,000 and ≈ 15,000) that reacted with a monoclonal anti-PRL receptor antibody and still specifically bound PRL, while the complete immunoreactive PRL binding unit (Mr ≈ 42,000) disappeared. Neither chymotrypsin nor V8 protease were able to generate any immunoreactive receptor fragments. These receptor fragments may represent smaller PRL binding receptor form(s) of biological significance.     

Prolactin-like hormone in the nematode Trichinella spiralis larvae

Expression of prolactin (PRL) or prolactin-like hormone has been reported in invertebrates. We investigated the larval phase of Trichinella spiralis: (a) to express 23 kDa PRL, (b) to define its localization and (c) to test its possible biological activity. Immunostaining in isolated larvae demonstrated positive material to 23 kDa PRL by all along the stichosome, specifically in the stichocytes. Homogenized immunoblot larvae showed a 23 kDa protein band. To assess PRL release and its biological activity, larvae were incubated in culture medium and the excretory/secretory products were analyzed by the Nb2 cells bioassay. A cellular growth equivalent until 10 nM PRL and using antibody against 23 kDa PRL, the growth was blocked. In conclusion our result provides evidence that PRL-like hormone is expressed and secreted by the larvae of T. spiralis.     

Mitogenic and binding properties of monoclonal antibodies to the prolactin receptor in Nb2 rat lymphoma cells. Selective enhancement by anti-mouse IgG

Three monoclonal antibodies (mAbs) (T6, U5, and U6) against prolactin (PRL) receptors in rat liver were studied in the rat lymphoma lactogen-dependent (Nb2-11C) and autonomous (Nb2-SP) cell lines. The mAbs had strong affinity for lactogen receptors (Ka = 12-14 nM-1), similar to that of human growth hormone (hGH) which is a lactogenic hormone. T6 and hGH competed for the same binding site, while U5 and U6 interacted with another epitope. The 125I-hGH-receptor complex could be immunoprecipitated by either U5 or U6, but not by T6. Affinity labeling and immunoblotting revealed that hGH and U6 bind to a protein of 63-65 kDa. T6, U5, and U6 were mitogenic in Nb2-11C cells but their respective potencies were 185-, 70-, and 4700-fold lower than that of hGH. Anti-mouse IgG enhanced the mitogenic effect of all three mAbs and almost completely abolished the differences between them, although their mitogenic activity was still 60-120-fold lower than hGH. Des-13-hGH, a competitive antagonist of hGH which hardly effected the binding of 125I-U5, inhibited the U5-stimulated proliferation of Nb2-11C cells in a noncompetitive manner, indicating that simultaneous binding of both ligands fixed the receptor in a nonactive conformation. A Fab fragment of T6 was not mitogenic, and inhibited the hGH-induced mitogenesis in a competitive manner, but its mitogenicity could be restored by anti-mouse IgG. We suggest that the dimerization or oligomerization of the lactogen receptor in Nb2-11C cells is an obligatory step in the transduction of the mitogenic signal. It may be induced by binding of the mAb to a site, which can be either identical or may even be distinct from that which binds the lactogenic hormone.     

Prolactin actions on casein and lipid biosynthesis in mouse and rabbit mammary gland explants are abolished by p-bromphenacyl bromide and quinacrine, phospholipase A2 inhibitors

p-Bromphenacyl bromide (BPB) at concentrations of 50 microM and above and quinacrine (50 microM) abolished the actions of prolactin (PRL) on casein and lipid biosynthesis in cultured mouse mammary gland explants. In cultured rabbit mammary gland explants, 100 microM BPB or quinacrine abolished the PRL stimulation of casein synthesis, while 50 microM BPB or 250 microM quinacrine abolished the PRL stimulation of lipid biosynthesis. Since BPB and quinacrine are known to inhibit the enzyme phospholipase A2 (PLA2), it is possible that ongoing PLA2 activity is essential for prolactin to express its actions on at least certain lactogenic processes.     

Effects of glucocorticoids on casein gene expression in the rabbit.

Milk protein synthesis is initiated by prolactin and a glucocorticoid. In the rabbit, prolactin alone is sufficient. However, glucocorticoids potentiate the action of prolactin. The stimulatory effect of glucocorticoids was evaluated after injections of hydrocortisone acetate alone or associated with prolactin by measurements of (a) the total RNA and DNA content of mammary glands, (b) the lactose synthetase activity, (c) casein synthesis, and (d) the concentration of casein mRNA in total cellular RNA and in polysomal RNA by hybridization with its cDNA. The glucocorticoid, totally inactive alone, proved to have a stimulatory effect proportional to the dose injected when prolactin was present. This effect was more evident with low doses of prolactin. Glucocorticoids proceeded by amplifying the capacity of prolactin to enhance the concentration of casein mRNA available for translation. A parallel effect of glucocorticoids on translation of casein mRNA was suspected. Glucocorticoids injected with low doses of prolactin were unable to mimic all the effects of high doses of prolactin alone.     

Mutational analysis of a lactogenic hormone reveals a role for lactogen-specific amino acid residues in receptor binding and mitogenic activity

Mutant forms of mouse placental lactogen-II (PL-II) have been generated to assess the role of specific amino acid residues and protein regions in binding to the PRL receptor and in mitogenic activity. Conversion of any of three lactogen-specific residues significantly reduced both of these hormone functions; mutation of the two other lactogen-specific amino acids revealed only minimal effects unless these changes were coupled with a second mutation. Deletions within the PL-II protein all resulted in a complete loss of function, but switching regions between PL-II and proliferin, another member of the prolactin family in the mouse, did yield a chimeric protein with some PRL-like activity. This activity was increased substantially by conversion of one amino acid residue in the proliferin region to the corresponding lactogen-specific residue. The locations of the amino acids that have been found to affect hormone function are predicted to be closely apposed in the folded protein, suggesting that this region may be the site of interaction of this lactogenic hormone with the PRL receptor.     

Effects of monoclonal antibodies on the function of acetylcholine receptors purified from Torpedo californica and reconstituted into vesicles

We tested the effects of 62 monoclonal antibodies (mAbs) to acetylcholine receptors from Torpedo californica on the function of receptor reconstituted into lipid vesicles. Two of these mAbs, mAbs 148 and 168, inhibited carbamylcholine-induced 22Na+ uptake into vesicles. The rate of 125I-labeled alpha-bungarotoxin (125I-alpha BGT) binding to the reconstituted liposomes was also reduced, although 125I-alpha BGT binding at equilibrium was not affected. Agonist-induced desensitization of the receptor was also affected by these mAbs. mAb 148 binds to the beta subunit of receptor, and mAb 168 binds to the gamma subunit. Both mAbs bind to the cytoplasmic surface of the receptor; correspondingly, both block function when added before reconstitution, and both were found to have no effect on function when added to preformed vesicles. Their effects were not due to interference with the reconstitution process. Both mAbs were capable of cross-linking receptors. In contrast to the bivalent mAbs, monovalent Fab fragments of these two mAbs had little effect on receptor function, which suggests that the effects of the bivalent mAbs resulted primarily from cross-linking receptors.     

Serum prolactin levels and prolactin binding activity in adrenals and kidneys of male rats after dehydration, salt loading, and unilateral nephrectomy.

Serum prolactin (PRL) levels and PRL binding activity in microsomal membranes from kidneys and adrenals were measured in control, water-deprived, unilaterally nephrectomized, and salt-loaded male rats. Unilateral nephrectomy and water deprivation increased serum prolactin levels significantly. Unilateral nephrectomy did not alter PRL binding activity in the kidneys, but significantly increased it in the adrenal glands. Salt loading had no effect on serum prolactin levels or PRL binding in the kidneys; but significantly increased PRL binding in the adrenal glands. Inhibition curves and tests of cross reactivity with LH, FSH, TSH, and GH showed that binding of PRL to its receptors in the kidneys and adrenals was specific. These observations suggest that PRL has a role in salt and water metabolism and that PRL receptors in the kidney and adrenals participate in this regulatory system.     

Effect of ketanserin, an inhibitor of 5-HT2 receptors, on the aldosterone-stimulating action of metoclopramide

To estimate the possible involvement of a peripheral serotonergic pathway in the mechanism of the aldosterone-stimulating effect of metoclopramide (M) the plasma aldosterone (PA), renin activity (PRA) and prolactin (PRL) response to M was studied in 6 normal subjects before and after administration of ketanserin (K), a pure, specific, and selective blocking agent of 5-hydroxytryptamine type 2 (5-HT2) receptors. With K preadministration the M-induced increase of PRL was similar to that observed in control conditions, in accordance with the specific and peripheral antiserotonergic action of the drug. K potentiated the PA and PRA elevation in response to M. These data suggest that the PA response to M is not related to M's agonist activity at the peripheral 5-HT2 receptors level. The results further indicate that K can induce an enhancement of the activity of renin-angiotensin-aldosterone system with an higher PRA and PA response to stimulatory action of M.     

Expression of the full-length rabbit prolactin receptor and its specific domains in baculovirus infected insect cells

The prolactin receptor is a membrane protein mainly involved in the development of the mammary gland and in lactation in mammals. We used specific cDNA constructs and the insect/baculovirus expression system and produced independently and in large amounts several recombinant forms of the rabbit mammary gland prolactin receptor: the full-length receptor (L1, L2), a truncated membrane form (S), a secretable form of the extracellular domain (E) and two forms of the intracellular domain (I1, I2). Of these forms, the L1 and L2 are associated with the membrane fraction, the E is predominantly secreted into the medium and the I1 and I2 are expressed as soluble proteins and surprisingly, a great portion accumulates in the culture medium. The molecular mass (94 kDa) of the expressed full-length receptor corresponds to the translation product of the entire cDNA coding region. The receptor biochemically identified in the rabbit mammary gland is however much shorter. Thus, in the mammary gland, the receptor presumably undergoes post-translational modifications. The receptor forms L1, L2 and S bind prolactin with specificity and affinity similar to those reported for the native receptor. They also interact with two monoclonal antibodies, M110 and A917, specific for the native conformation of the hormone-binding site. The I1 and I2 forms do not bind prolactin, whereas the E form does. Thus, the hormone binding site is located in the extracellular domain which can function autonomously as a PRL-binding soluble protein. However, the E form binds prolactin with a higher affinity than the native receptor and it does not bind one of the two antireceptor monoclonal antibodies, known to be hormone binding-site specific. Thus, the conformation of the native receptor and that of the E form differ.     

Observation of production of immunoactive prolactin by normal human connective tissue in cell culture

Summary Data from our in vitro studies indicate a new source of prolactin (PRL)-like activity, normal human connective tissue. Fascial cells from primary culture and subsequent passages produced an extracellular antigen which specifically reacted in a radioimmunoassay RIA developed to detect human pituitary PRL. An initial peak of first surge of fascial PRL-like activity occurred between 4 and 15 d in primary culture. Ibuprofen, cytotoxic levels of 0.01% azide, or 7.5 mM EDTA and medium lacking serum [fetal bovine serum (FBS)] significantly (P≤0.05) reduced PRL-like activity levels, whereas female steroids, 257 to 342 milliosmolarity, 1 to 3.6 mg/ml glucose, 2 to 20% FBS, and dialyzed FBS (MWCO ⊂) 1 kDa) were without effect. Optimum production of PRL-like activity occurred at pH 7.3. A second surge began after 18 d and continued until passage indicating that perhaps two populations of cells produced PRL-like activity in primary culture. Production of PRL-like activity by cells from early passages (1 and 2) became detectable at confluence, was serum-dependent, showed two patterns (tonic, rising to plateau), and averaged 3.2 fg·cell⁻¹·3 d⁻¹ feed interval. Cells from late passages showed morphologic damage from repetitive trypsinization, aging, and reduced production of PRL-like activity with aberrant production pattern. Production of PRL-like activity was maintained in an unusual long-term culture. these in vitro studies demonstrate the most recently recognized and ubiquitous source of human extrapituitary PRL or PRL-like activity, normal connective tissue (fascia). A portion of this paper was presented at the 33rd Annual Meeting of the Society for Gynecologic investigation, Toronto, Ontario, Canada, 19–22 March 1986. This work was supported in part by grant HD21883 (to D. H. R.) from the National Institutes of Health, Bethesda, MD.