Purification and partial sequence of the rabbit mammary gland prolactin receptor

1. The prolactin receptor from rabbit mammary gland was purified to near homogeneity using a novel hydrophobic interaction chromatographic procedure. 2. Part sequencing (101 residues) revealed 34% identity with the rabbit liver growth hormone receptor, providing support for the existence of a new class of transmembrane receptors regulating growth and lactation.     

Early effects of prolactin on lactating rabbit mammary gland

Summary Effects of prolactin on the secretion of milk proteins have been investigated by incubating mammary tissue fragments from lactating rabbits. Within 15min of adding the hormone to the incubation medium, cell morphology is modified: the relative volume occupied by the Golgi region is greatly increased. When prolactin is added immediately after a pulse labelling of proteins (3 min with ³H-L-leucine), the amount of labelled caseins secreted during one hour is significantly increased. This increase proceeds neither from an acceleration of intracellular transit of caseins (as shown by electron microscopic autoradiography) nor by an enhancement of amino acid uptake (as measured by incorporation of non-metabolizable amino acids) nor by an increase of overall protein synthesis, during the first hour.The action of prolactin on the morphology of such subcellular organelles as the Golgi apparatus and its influence on casein secretion are discussed.We are extremely grateful to Professor Hubert Clauser for his helpful advice. We thank Mrs. Georgette Gianni for her excellent technical assistance and Miss Aline Solari for her contribution to statistical analysis     

Two separate receptors for prolactin in the rabbit mammary gland

Rabbit mammary gland PRL receptors in the microsome fraction were solubilized with the zwitterionic detergent Chaps, and were separated into two fractions (Fr. A and B) by ion-exchange chromatography. The number of receptors in Fr. B was about 2.2 times greater than in Fr. A. In sucrose gradient centrifugation analysis, PRL receptors in Fr. A and Fr. B sedimented at different positions. After binding 125I-PRL, the apparent molecular weight (mol wt) of the PRL receptor in Fr. A changed from 42,400 to 65,500 and that in Fr. B changed from 89,400 to 108,000, suggesting that each binding subunit interacts with one PRL molecule. Cross-linking 125I-PRL to receptors revealed little change following SDS-PAGE, in the autoradiogram patterns of the microsome PRL receptors, either in the presence or absence of dithiothreitol. Both the microsome and the Chaps extract contained two major binding subunits (mol wt, 83,200 and 36,800) and one minor subunit (mol wt, 20,800). The mol wt of the dominant PRL receptors in Fr. A and Fr. B were 36,800 and 83,200, respectively. The latter form did not dissociate into a 36,800 mol wt form, suggesting that the rabbit mammary gland contains two independent binding subunits with mol wt of 36,800 and 83,200. Data showed that PRL receptors in the rabbit mammary gland are mostly the high Kd type receptor with a mol wt of 83,200.     

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.     

Interaction between prolactin and rabbit mammary prolactin receptor in the presence of environment-modifying agents

The binding assay of prolactin (PRL) to the receptor in the rabbit mammary gland was carried out with varying concentrations of NaCl, KCl, CaCl2, MgCl2, glycerol, glucose, sucrose and urea. The agents did not affect the binding capacity. The ionic bond-breaking agents (NaCl and KCl) had little effect on changes in the association rate constant (k+1) of PRL binding to the receptor and the dissociation rate constant (k-1) of bound PRL. The inclusion of other agents changed the k+1 and the k-1. Among the agents examined, chaotropic salts (CaCl2 and MgCl2) inhibited the binding of PRL greatly, and were the most effective in decreasing the k+1. Both hydrogen- and hydrophobic bonds are involved in the interaction between PRL and the receptor. The data suggest that hydrophobic bonding is primarily an important force participating in the binding of PRL to its receptor.     

Immunochemical characterization of casein from rabbit mammary gland.

1. Excellent precipitating antibodies to rabbit recombined casein polypeptides were obtained in a sheep after 8 weeks of immunization with rabbit recombined polypeptides coupled to Sepharose-albumin. 2. The antiserum was assessed for specificity by several immunochemical techniques and was monospecific when tested against acid-precipitated casein, recombined casein and extracts of lactating rabbit mammary tissue. 3. A specific anti-casein immunoglobulin fraction was prepared by immunoadsorption of the antiserum by using Sepharose-recombined casein as immunoadsorbent. 4. The specific anti-casein immunoglobulin was used to prepare a Sepharose-anti-casein immunoadsorbent for the isolation of casein from extracts of rabbit mammary tissue.     

Biological activities of binding site specific monoclonal antibodies to prolactin receptors of rabbit mammary gland

The biological activity of three monoclonal antibodies (mAbs) against the rabbit mammary prolactin (PRL) receptor (M110, A82, and A917) were investigated using explants of rabbit mammary gland. The three mAbs which were all able to inhibit the binding of 125I-ovine prolactin to its receptor had different biological activities. Two mAbs (M110 and A82) were able to prevent the stimulating effect of PRL on casein synthesis when the molar ratio between the mAb and PRL was 100. At a lower concentration, M110 moved the PRL dose-response curve to the right by a factor of 2.4. This mAb was also effective in vivo, reducing milk production in a lactating rabbit, in a similar fashion to the prolactin lowering drug, CB-154. One mAb (A917) was able to mimic the action of PRL on both casein and DNA ([3H]thymidine incorporation) synthesis, whereas the other two mAbs were without any stimulatory effect. For this stimulatory effect to be observed, bivalency of the antibody was essential, since monovalent fragments, which were able to inhibit PRL binding, had no agonistic activity. The ability of the mAbs to induce a down-regulation of receptors was also studied. M110, which was equipotent to PRL in occupation of receptors, induced no down-regulation, while A917, which had full biological activity, induced only a small degree of down-regulation. These studies suggest that the binding domain of the receptor might be relatively complex, since only a part of this domain recognized by the antibody with PRL-like activity was able to induce hormonal action. Alternatively, only those antibodies able to microaggregate the receptors may possess PRL-like activity.     

Novel multialveolar epithelial structures from rabbit mammary gland that synthesize milk specific fatty acids in response to prolactin

Summary Multialveolar mammary epithelial structures have been prepared from rabbit mammary gland by treating the tissue with collagenase plus hyaluronidase. These structures synthesize milk specific fatty acids when cultured with physiological concentrations (0.05 μg/ml) of prolactin in the presence of insulin and corticosterone. They have many of the advantages but few of the disadvantages of either mammary explains or primary cells in culture. For example, they are easily prepared in large numbers and respond to prolactin in culture even in the absence or other tissue extracts. Because their level of organization is intermediate between that of explants and single cells, they provide a complementary system for studies on mammary differentiation. This work was supported by grants from the Agricultural Research Council of Great Britain and the Royal Society to R. R. Dils, by U.S. Public Health Service Grant CA-16392, and an American Cancer Society-Eleanor Roosevelt International Cancer Fellowship to H. L. Hosick. Prolactin was a gift from the Endocrinology Study Section, National Institutes of Health, Bethesda, MD.     

Novel multialveolar epithelial structures from rabbit mammary gland that synthesize milk specific fatty acids in response to prolactin

Multialveolar mammary epithelial structures have been prepared from rabbit mammary gland by treating the tissue with collagenase plus hyaluronidase. These structures synthesize milk specific fatty acids when cultured with physiological concentrations (0.5 micrograms/ml) of prolactin in the presence of insulin and corticosterone. They have many of the advantages but few of the disadvantages of either mammary explants or primary cells in culture. For example, they are easily prepared in large numbers and respond to prolactin in culture even in the absence of serum or other tissue extracts. Because their level of organization is intermediate between that of explants and single cells, they provide a complementary system for studies on mammary differentiation.     

Cyclosporin A and rabbit mammary prolactin receptors

The binding of cyclosporin A and ovine prolactin to rabbit mammary gland membranes was determined. CsA bound with a Kd of 2.2 X 10(-6)M whereas prolactin bound with a Kd of 2 X 10(-10)M. The binding of each ligand was an independent event and neither ligand influenced the binding of the other ligand showing that CsA does not inhibit the binding of prolactin to its specific receptor in this system.     

Hydrolysis of prolactin by a serine proteinase from mammary gland secretory cell mitochondria]

Mitochondrial proteinase isolated from secretory cells of the mammary gland of lactating rats able to hydrolyze 125I-labeled and native prolactin (PRL) has been studied. The enzyme represents a serine proteinase and is localized in the inner mitochondrial membrane. The molecular mass of the enzyme is 17-18 kDa, pH optimum is at 8.0-9.0. Partial purification of the enzyme has been carried out. The Km constant for 125I-PRL is equal to 10(-6) M, that for 2% hemoglobin is 1.2 x 10(-4) M. Analysis of products of rat and ovine PRL hydrolysis by proteinase using high performance liquid chromatography and PAAG electrophoresis revealed the formation of large-size fragments of the hormone. A possible role of proteinase in the mechanism of PRL action on mammary gland tissues is discussed.