STUDIES ON PITUITARY LACTOGENIC HORMONE : III. SOLUBILITIES OF SHEEP AND BEEF HORMONES

The solubility of sheep pituitary lactogenic hormone in 0.302 M NaCl at pH 2.02 (solution in HCl) has been determined at room temperature. It showed a constant solubility in the presence of a considerable excess of the solid phase, an indication that the preparation contained but one component. Beef lactogenic hormone showed a constant solubility in distilled H₂O at 7–8°C in the presence of excess of the solid phase. The salting-out effect of NaCl in acid solution of both beef and sheep hormones has been studied at room temperature. In these studies both preparations behaved as pure substances, but they exhibited differences in solubility, thus indicating a species specificity.     

A new microbioassay for the measurement of lactogenic hormones in human serum

The standard Nb2 assay for biologically active prolactin has been modified to allow a rapid convenient microbioassay without loss of specificity or accuracy. Lactogenic hormones specifically stimulate the replication of Nb2 node rat lymphoma cells in suspension culture and form the basis of a currently available bioassay to measure prolactin and growth hormone in human serum. A new microbioassay was developed using microtest plates enabling a large number of samples to be assayed simultaneously whilst maintaining the overall sensitivity of the bioassay for lactogenic hormones. Growth of the Nb2 node lymphoma cells, measured by a light scattering technique using optical density on a spectrophotometer, was shown to be closely correlated with the cell number determined on a Coulter counter. Addition of excess anti-human prolactin and anti-human growth hormone completely inhibited the growth stimulatory effects of both human prolactin and human growth hormone. This new microbioassay (BA) and conventional radioimmunoassay (RIA) were used to measure lactogenic hormones in 48 normal subjects. There was a close correlation between the results of both assays for each hormone studied in the control sera. The mean basal BA/RIA ratio was 1.5 (range 0.8-2.0) for prolactin, 0.7 (range 0-4.5) for growth hormone and 1.3 (range 0.5-1.9) for total lactogenic activity.     

STUDIES ON PITUITARY LACTOGENIC HORMONE : I. ELECTROPHORETIC BEHAVIOR

The Tiselius technique of electrophoresis has been used to study the homogeneity and mobility of pituitary lactogenic hormone. The lactogenic preparation employed by us shows only one sharp band in schlieren photographs, indicating a high degree of homogeneity. From the determination of the mobility of the hormone at different hydrogen ion concentrations in acetate and phosphate buffer of ionic strength 0.055, the isoelectric point was found to be 5.70 and the See PDF for Equation value 4.5 x 10^–5. The difference in the mobility reported for White''s crystalline preparation and that found with our high potency lactogenic preparation is discussed.     

Methionine oxidation in human growth hormone and human chorionic somatomammotropin. Effects on receptor binding and biological activities

The oxidation of the methionine residues of human growth hormone (hGH) and human chorionic somatomammotropin (hCS) to methionine sulfoxide by hydrogen peroxide has been studied. The kinetics of oxidation of individual methionine residues has been measured by reverse-phase high pressure liquid chromatography tryptic peptide mapping. Met-170 is completely resistant to oxidation in both hormones. The other 3 methionine residues in hCS (Met-64, Met-96, and Met-179) have markedly different reaction rates. Oxidation of the methionine residues does not appear to cause gross conformational changes in either hGH or hCS, as judged by CD and 1H NMR spectroscopy. Oxidation of Met-14 and Met-125 in hGH has little effect on affinity of the hormone for lactogenic receptors or on its potency in the Nb2 rat lymphoma in vitro bioassay for lactogenic hormones. The oxidation of Met-64 and/or Met-179 in hCS reduces profoundly both its affinity for lactogenic receptors and its in vitro biological potency. It is inferred by induction that residues 64 and/or 179 are critical for the binding of both hGH and hCS to lactogenic receptors and the expression of lactogenic biological activity.     

Epidermal growth factor receptor, platelet-derived growth factor receptor, and c-erbB-2 receptor activation all promote growth but have distinctive effects upon mouse mammary epithelial cell differentiation.

Three different receptor tyrosine kinases, epidermal growth factor (EGF), c-erbB-2/neu, and platelet-derived growth factor (PDGF) receptors, have been found to be present in the mouse mammary epithelial cell line HC11. We have investigated the consequences of receptor activation on the growth and differentiation of HC11 cells. HC11 cells are normal epithelial cells which maintain differentiation-specific functions. Treatment of the cells with the lactogenic hormones glucocorticoids and prolactin leads to the expression of the milk protein beta-casein. Activation of EGF receptor has a positive effect on cell growth and causes the cells to become competent for the lactogenic hormone response. HC11 cells respond optimally to the lactogenic hormone mixture and synthesize high levels of beta-casein only if they have been kept previously in a medium containing EGF. Transfection of HC11 cells with the activated rat neuT receptor results in the acquisition of competence to respond to the lactogenic hormones even if the cells are grown in the absence of EGF. The activation of PDGF receptor, through PDGF-BB, also stimulates the growth of HC11 cells. Cells kept only in PDGF do not become competent for lactogenic hormone induction. The results show that activation of the structurally related EGF and c-erbB-2/neu receptors, but not the PDGF receptor, allows the HC11 cells to subsequently respond optimally to lactogenic hormones.     

Features of structure-activity organization of prolactin

The secondary structure of seven hormones of the prolactin family was predicted by two known prediction algorithms with the following averaging of the results for the whole homologous group of proteins. It was shown that the mentioned hormones are related to the alpha-helical type of protein molecules. The comparative analysis of the prolactin family and the kindred growth hormone family has been carried out on different levels of their structural organization. A conclusion is drawn, that despite the significant differences in the primary structures and small differences in the secondary structures, the three-dimensional structure for the prolactin molecules and the growth hormone are very similar and repeat in the same way the packing of alpha-helices. The study of the relationship of the prolactin structure and function has been carried out. The regions of the amino acid sequence, able to form prolactin antigenic sites and conditionally incorporated into two highly specific spatial groups were revealed. The region of the primary structure 80-137 determining lactogenic and proliferational function of the molecule and forming the alpha-hairpin in the tertiary structure has been discovered. The structural particuliarity of one of the binding sites of prolactin and human growth hormone with lactogenic receptor was reveal. An explanation for the absence of lactogenic activity in all kinds of growth hormones except human ones has been proposed.     

Homology modeling of rabbit prolactin hormone complexed with its receptor

The pituitary hormone prolactin (prl) is implicated in a number of biological functions, especially lactation, which is mediated through specific lactogenic receptors (PrlR). Human growth hormone (hGH) is also a pituitary hormone responsible for linear growth. While the growth hormone receptor (hGHR) binds only hGH, hPrlR can interact with both hGH and hPrl. Using structural information from the human growth hormone (hGH)/receptor (hGHR) complex, we modeled by homology a complex between rabbit prolactin hormone (rbPrl) and its receptor (rbPrlR). While the somatogenic hormone/somatogenic receptor (hGH/hGHR) and somatogenic hormone/lactogenic receptor (hGH/hPrlR) interactions are now known and well studied, here we propose a model for the interaction of the lactogenic hormone with its receptor (rbPrl/rbPrlR), and compare these three kinds of ligand/receptor interaction. We identified residues contributing to the active site and tested the potential dimerization of the receptor. Biochemical studies and information deduced from the modeled complex do not exclude a homodimeric form but point to a functional heterodimeric complex. Proteins 27: 459–468, 1997. © 1997 Wiley-Liss, Inc.     

Milk protein expression and ductal morphogenesis in the mammary gland in vitro: hormone-dependent and -independent phases of adipocyte-mammary epithelial cell interaction

Epithelial cell differentiation frequently occurs in situ in conjunction with supporting mesenchyme or connective tissue. In embryonic development the importance of the supporting mesenchyme for cytodifferentiation and morphogenesis has been demonstrated in several epithelial tissues, but the importance of epithelial-connective tissue interactions is less well studied in adult epithelial organs. We have investigated the interaction of adult mammary epithelial cells with adipocytes, which compose the normal supporting connective tissue in the mammary gland. Mammary epithelial cells from mice in various physiological states were cultured on cellular substrates of adipocytes formed from cells of the 3T3-L1 preadipocyte cell line. We found that there were two distinct phases to the interaction of epithelial cells with adipocytes. Cytodifferentiation of the epithelial cells and milk protein production were dependent on lactogenic hormones (insulin, hydrocortisone, and prolactin), whereas ductal morphogenesis was lactogenic hormone independent. When cultured on preadipocytes or adipocytes, mammary epithelial cells from never pregnant, pregnant, lactating, and involuting mice responded to lactogenic hormones rapidly by producing and secreting large amounts of alpha-, beta-, and gamma-casein and alpha-lactalbumin. This response was seen in individual as well as in clusters of epithelial cells, but was not seen if the same cells were cultured on tissue culture dishes without adipocytes, on fibroblasts (human newborn foreskin fibroblasts) or in the presence of adipocytes but in the absence of lactogenic hormones. Continued incubation of mammary epithelial cells on adipocytes in the presence or absence of lactogenic hormones resulted in the formation of a branching ductal system. Mammary epithelial cells in ducts that formed in the absence of lactogenic hormones produced no casein, but rapidly synthesized casein when subsequently exposed to these hormones. Ultrastructural studies revealed that the formation of a basement membrane occurs only in co-cultures of mammary epithelium with adipocytes or preadipocytes. Ultrastructural changes associated with secretion occurred only in the presence of lactogenic hormones. We propose that growth and formation of a ductal system in vitro can occur in the absence of lactogenic hormones, but that certain environment-associated events must occur if the epithelium is to become responsive to lactogenic hormones and undergo the cytodifferentiation associated with lactation.     

Zinc binding to human lactogenic hormones and the human prolactin receptor

Zinc half sites are present in all human lactogenic hormones: human prolactin (hPRL), growth hormone (hGH), placental lactogens (hPL) and the hPRL receptor (hPRLr). The influence of divalent zinc (Zn(2+)) as measured by intrinsic fluorescence or FRET in each of these hormones is unique and is affected by the presence of varying stoichiometries of hPRLr. These data show that both Zn(2+) and hPRLr binding influence hPRL conformers in an interdependent fashion. Although each of these three lactogenic hormones bind hPRLr and induce a biological response that is sensitive to the presence of increasing concentrations of Zn(2+), each hormone is unique in the mechanistic details of this process.     

Ovine prolactin and human growth hormone derivatives. Specific modification of their alpha-amino groups

The alpha-amino group of ovine prolactin (oPRL) and human growth hormone (hGH) was selectively modified by transamination with glyoxylic acid. No difference was found in the binding capacity of transaminated oPRL to rat liver lactogenic receptors with respect to its control, although both samples showed a decrease in its binding capacity with reference to the native hormone. This decrease was due to conformational changes caused by the reaction conditions and not by the transamination itself, as shown by the circular dichroism spectra. Transaminated hGH retained the full binding capacity of the hormone. These results suggest that the alpha-amino group is not relevant for the binding to lactogenic liver receptors in both lactogenic hormones.     

Alanine-scanning mutagenesis of human prolactin: importance of the 58-74 region for bioactivity.

We have generated 10 alanine mutants of human PRL (hPRL), a member of the PRL/GH family, to investigate the involvement of the highly conserved 58-74 region in the biological behavior of the protein. When treated with polyclonal anti-hPRL antibodies, all mutants were immunologically indistinguishable from the unmodified hPRL, and circular dichroism analyses indicated that their alpha-helix content was similar to that of the unmodified hormone. Mutations C58A, K69A, and, to a lesser extent, P66A affected drastically the ability of hPRL first to bind to the lactogenic receptor and second to stimulate the proliferation of Nb2 lymphoma cells, proving the importance of the 58-74 peptide segment for hPRL bioactivity. Binding affinities of these mutants to the Nb2 lactogenic receptor have been compared to lactogenic binding data previously obtained for several mutants of hGH. The comparison reveals that the residues involved in the biological properties of the two proteins are not at topologically equivalent positions. Hence, we suggest that the binding of these hormones to the lactogenic receptors occurs through a different molecular mechanism having distinct requirements at the residue level.     

Simultaneous isolation of prolactin and growth hormone from "discarded acid pellet" obtained from buffalo pituitaries

An acid pellet, obtained as a side fraction from a conventional gonadotropin purification pathway, has been found to contain the bulk of the pituitary lactogenic hormones (growth hormone or GH and prolactin or PRL). This discarded side fraction has been utilized to obtain buffalo lactogenic hormones (buGH and buPRL), simultaneously, and in bulk. The immunoreactivities of the purified semi-pure buffalo GH and PRL (APECS and APP-I, respectively) preparations were compared by direct binding ELISA with semi-pure standard buGH and PRL (ECS and EP-I, respectively) and were found to be as pure as standard semi-pure buGH and buPRL. When checked by direct binding ELISA using buGH and buPRL antisera, it was observed that APECS and APP-I were not cross-immunoreactive. SDS-PAGE and western blot analysis of APECS and APP-I showed major bands located at the same positions as in the case of standard semi-pure preparations (20 kDa for APECS and 23 kDa for APP-I). The semi-purified buGH and buPRL (APECS and APP-I) were converted to a highly purified preparation by chromatographing them via Sephacryl S-200 gel-filtration chromatography.     

THE MOLECULAR EVOLUTION OF PITUITARY HORMONES

1. The pituitary hormones can be divided into 4 families; within each the members are structurally related and have probably evolved from a common ancestor by a process of gene duplication and divergence. 2. Recent structural studies have revealed much about the evolution of proteins. The roles of point mutation, gene duplication and partial gene duplication in molecular evolution have been highlighted, and the nature of the evolutionary forces involved has been extensively debated. The information available about the evolution of proteins in general provides a background for consideration of pituitary hormone evolution. 3. The structure and function of the mammalian neurohypophysial hormones (oxytocin and vasopressin) has been studied in detail. Related (structurally similar) peptides have been found in the neurohypophyses of lower vertebrates and have been Characterized in many instances. Several schemes have been proposed for the evolution of these hormones. 4. The vasopressins of the pig and its relatives show a genetic polymorphism. The roles of neurohypophysial hormones in lower vertebrates are very varied and not fully understood. 5. The ACTHs and MSHs are members of a second family of pituitary hormones. They are polypeptides of moderate size. Studies on amino-acid sequences have been carried out for ACTHs and MSHs from several mammals. α-MSH is identical in all cases studied in detail, but β-MSH and ACTH vary to some extent. There is considerable sequence homology between the hormones in this family - indicating a common phylogenetic origin and several gene duplications. 6. Dogfish MSH is the only non-mammalian hormone of the ACTH-MSH family to have been studied in detail. Two MSHs have been isolated from this species; both resemble the a-MSH of mammals in amino-acid sequence. ACTH-like and MSH-like hormones exist in many other vertebrate groups, but have not been characterized fully. 7. Structure-function relationships have been widely studied in the ACTH-MSH family, and have some interesting evolutionary implications. Polymorphism of P-MSHs is found in some mammals. 8. A third family of protein hormones includes pituitary prolactin and growth hormone, and placental lactogen. These are proteins of moderate size which have been shown to be widely distributed among the vertebrates. Species specificity can be recognized with regard to biological, immunological and structural properties. 9. Amino-acid sequences have been determined for growth hormones and prolactins from several mammals. There is sequence homology between growth hormone and prolactin. Human placental lactogen closely resembles human growth hormone. A phylogenetic tree has been constructed for this protein family. Rates of evolution within the group are rather variable. 10. The fourth family of pituitary hormones (FSH, LH, TSH and some related placental hormones) are all glycoproteins and have a subunit structure. Extensive sequence studies have been carried out on the hormones from some mammals, and show that there is considerable homology between the various subunits. The α-subunits of human TSH, LH and HCG (and probably FSH) are very similar. The β-subunits are different, but homologous. Evolution of this family clearly took place by a series of gene duplications followed by gene divergence. Schemes whereby this could have occurred have been discussed. Related hormones occur in lower vertebrates, but have not been fully characterized. Some lower vertebrates may possess only one gonado-trophin. 11. The pituitary hormones provide an interesting range of evolutionary problems, and are useful models for the study of molecular evolution. The evolutionary processes involved in their diversification have been discussed, with particular reference to the co-evolution of hormones and their receptors. Neutral mutations and gene duplications may have played a role in providing co-existing variation of hormones and receptors. 12. A speculative model for the evolution of neurohypophysial hormones is proposed, as an example of how molecular evolution may have operated in this and other hormone groups. 13. Homologies have been proposed between the various families of pituitary hormones, and between pituitary proteins and other entero-secretory proteins. The pituitary protein hormones were probably elaborated from smaller molecules by a process of partial gene duplication.     

Serum prolactin and growth hormone determined by radioimmunoassay and a two-site immunoradiometric assay: comparison with the Nb2 cell bioassay

Serum levels of the two lactogenic hormones prolactin (PRL) and growth hormone (GH) were compared when determined by radioimmunoassay (RIA) and two-site immunoradiometric (IRMA) assays in 83 normal premenopausal women. The mean values for the PRL and GH results determined by RIA were higher than those obtained by IRMA, despite strong correlations between the two (PRL, r = 0.92; GH, r = 0.79). The lactogenic hormones were also determined together by the Nb2 cell bioassay (BA) in 38 of these same women, and the results compared with the sum of the PRL and GH immunoassays. There was a strong correlation between the BA and RIA (r = 0.75), and the BA/PRL+GH RIA ratio averaged 1.6 +/- 0.5. Corresponding values for IRMA were r = 0.66, and BA/PRL + GH IRMA 3.3 +/- 1.1. Thus, the polyclonal RIA antisera appeared to recognize bioactive hormone components not determined by the double monoclonal antibody IRMA. Another 23 women at risk for familial breast cancer, and 14 cystic breast disease patients were also studied. High BA, but normal RIA results, giving mean ratios of 2.4 +/- 1.1 and 3.6 +/- 3.0 respectively, suggest the presence of a further variant with high bioactivity not detected by RIA in these two clinical situations.     

Pituitary responsiveness to gonadotrophin-releasing and thyrotrophin-releasing hormones in children receiving phenobarbitone.

The effect of long-term treatment with phenobarbitone on pituitary responsiveness to gonadotrophin-releasing hormone and thyrotrophin-releasing hormone was studied in 20 boys being treated with the drug to prevent febrile convulsions. Baseline concentrations of luteinising and follicle-stimulating hormones were reduced as well as the responses of these hormones to stimulation with gonadotrophin-releasing hormone. Baseline prolactin concentrations were raised in comparison with those in normal children. The response of prolactin to thyrotrophin-releasing hormone, however, was impaired only in the children who had been receiving the drug for a long time. Phenobarbitone had no effect on the secretion of growth hormone. Further studies should be carried out to ascertain how long these effects on pituitary function last after phenobarbitone is withdrawn and whether this interference with pituitary function modifies the child''s subsequent development.     

Studies on pituitary lactogenic hormone. The primary structure of the porcine hormone.

The complete amino acid sequence of porcine lactogenic hormone has been elucidated. It consists of 199 amino acid residues with three disulfide bridges formed by residues 4-11, 58-174, and 191-199. The two tryptophan residues are in positions 91 and 150. A high degree of homology exists between the known structure of ovine prolactin and the porcine lactogenic hormone. This led to a re-examination of residues 82-90 in the ovine prolactin structure, where an extra leucine was found in position 88.     

A comparison of lactogenic receptors from rat liver and Nb2 rat lymphoma cells by using cross-linking techniques.

Lactogenic receptors were analysed with the use of the cross-linking agent disuccinimidyl suberate to attach covalently 125I-labelled ovine prolactin or human growth hormone to binding sites from (1) liver from pregnant rats and (2) the rat-derived Nb2 lymphoma cell line. Analysis by SDS/polyacrylamide-gel electrophoresis of the proteins cross-linked to labelled hormone in rat liver indicated a major specifically-labelled complex with an Mr of 68,000-72,000, when run under reducing or non-reducing conditions. With Nb2 cells a major specifically-labelled complex with an Mr of 97,000-110,000 was identified, but only when electrophoresis was run using reducing conditions. Assuming one hormone molecule (Mr 22,000-24,000) per hormone-receptor complex, then the receptor proteins have an Mr of 44,000-50,000 for rat liver and 73,000-88,000 for the Nb2 cells. For both cell types the receptors were of lactogenic specificity; lactogenic hormones competed for binding whereas somatogenic hormones did not. These studies suggest that the lactogenic receptors in rat liver membranes and Nb2 cells differ in two respects. Firstly, the Mr of the labelled receptor protein in Nb2 cells is greater than that of the corresponding receptor protein in rat liver membranes; secondly, the Nb2 cell receptor appears to exist as a disulphide-linked oligomer whereas the receptor in rat liver membranes does not.     

Lactogenic response of mouse mammary explants from different days of pregnancy to placental lactogen and pituitary prolactin

The lactogenic response of mouse mammary gland explants to human placental lactogen (hPL) and ovine pituitary prolactin (oPRL) was examined on days 10 to 18 of pregnancy by measuring 3H-amino acid incorporation into calcium-rennin precipitable casein. To determine the lactogenic response of the explants, the mean slopes of dose-response curves were calculated for each hormone treatment. Slope means of dose-response curves for oPRL and hPL did not differ from each other on any day of pregnancy examined. A triphasic pattern of response was suggested when slope means of dose-response curves for both hormones were plotted as a function of day of gestation. Peak responses were observed on days 10, 13 and 17-18. Combinations of oPRL and hPL, in ratios of oPRL:hPL = 2:1 and oPRL:hPL = 1:2, also produced a triphasic pattern of sensitivity very similar to that produced by either hormone alone. These results suggest that mouse mammary explants may be more sensitive to oPRL and hPL on days 10, 13 and 17-18 of pregnancy.     

Effects of gonadectomy on polymorphism in stored and circulating gonadotropins in the bullfrog, Rana catesbeiana. II. Gel filtration chromatography

Marked polymorphism was revealed in both stored and circulating forms of immunoreactive follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the bullfrog, Rana catesbeiana, by exclusion chromatography on columns of Sephracyrl-S200. FSH behaved as a more homogeneous and larger molecule than LH from the same pituitary or plasma, but the properties of both hormones in the plasma were markedly affected by gonadectomy. Chromatographic profiles of FSH stored in the pituitaries were similar in intact and gonadectomized frogs, but pituitary LH in the latter was comprised of a larger proportion of early eluting activity. Previously purified preparations of bullfrog FSH and LH were more homogeneous than these extracts. Differences between pituitary hormones in intact and gonadectomized frogs were small compared with those between circulating hormones. Plasma FSH and lH from gonadectomized frogs behaved as more homogeneous and larger molecules than those from intact frogs in which plasma gonadotropins were elevated normally or by injections with gonadotropin releasing hormone (GnRH). Some differences in circulating hormones were also observed between a normal male and female and both differed from gonadectomized an GnRH-treated intact frogs. Chromatographs of plasma gonadotropins in GnRH-treated animals generally resembled those of the hormones stored in the pituitary, whereas plasma FSH and LH in gonadectomized frogs appeared more homogeneous and larger than the pituitary-stored forms. Those pronounced differences in chromatographic properties of gonadotropins in intact and gonadectomized frogs correlate with previously observed effects of gonadectomy on clearance profiles of circulating FSH and LH.