Obligate Ordered Binding of Human Lactogenic Cytokines

Class 1 cytokines bind two receptors to create an active heterotrimeric complex. It has been argued that ligand binding to their receptors is an ordered process, but a structural mechanism describing this process has not been determined. We have previously described an obligate ordered binding mechanism for the human prolactin/prolactin receptor heterotrimeric complex. In this work we expand this conceptual understanding of ordered binding to include three human lactogenic hormones: prolactin, growth hormone, and placental lactogen. We independently blocked either of the two receptor binding sites of each hormone and used surface plasmon resonance to measure human prolactin receptor binding kinetics and stoichiometries to the remaining binding surface. When site 1 of any of the three hormones was blocked, site 2 could not bind the receptor. But blocking site 2 did not affect receptor binding at site 1, indicating a requirement for receptor binding to site 1 before site 2 binding. In addition we noted variable responses to the presence of zinc in hormone-receptor interaction. Finally, we performed Förster resonance energy transfer (FRET) analyses where receptor binding at subsaturating stoichiometries induced changes in FRET signaling, indicative of binding-induced changes in hormone conformation, whereas at receptor:hormone ratios in excess of 2:1 no additional changes in FRET signaling were observed. These results strongly support a conformationally mediated obligate-ordered receptor binding for each of the three lactogenic hormones.     

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.     

Assay of lactogenic hormones using receptors isolated from rabbit liver.

Using 125-I-labelled ovine prolactin and receptors isolated from the livers of rabbits, a sensitive method has been developed suitable for the assay of ovine, bovine, porcine, human and rat prolactins. These hormones showed competitive displacement of 125-I-ovine prolactin which was in general agreement with their respective activities in the pigeon crop sac bioassay. Human and monkey growth hormones and human placental lactogen, which have marked prolactin-like actions on mammary tissue were also effective competitors. Non-primate growth hormones (ovine, bovine, equine and canine) which do not have prolactin-like activity gave little if any displacement as did human FSH, LH, TSH, ACTH and bovine insulin. Preparations of equine and canine prolactin of varying purity gave dose-response curves although their activity as competitors relative to ovine prolactin was poor and not related to their pigeon crop stimulating activity. This indicates species differences between prolactins in hormone-receptor interaction. Experiments with antiserum to human growth hormone have suggested an effective method of making the assay specific in species such as man in which prolactin is not the sole hormone with lactogenic activity.     

Human growth hormone site 2 lactogenic activity requires a distant tyrosine164.

Comparison of crystallographic structures of human growth hormone, either bound to the prolactin receptor or free of receptors, reveals that human growth hormone binding to the prolactin receptor at site 1 is associated with a structural change in human growth hormone that influences the organization of residues which constitute site 2. We have identified Tyr164 as a residue that is critical for the propagation of this structural rearrangement. Tyr164 is a structural epitope for site 1 and is distal to site 2. Mutation of Tyr164 to glutamic acid (Y164E) does not affect the somatotrophic activity, absorption or fluorescence spectra or binding to the human prolactin receptor when compared to wild-type human growth hormone, indicating the subtle effects of the mutation. Lactogenic assays using extended concentrations of Y164E human growth hormone produce dose-response curves that are characterized by a right-shifted agonist phase and an unchanged antagonist phase when compared to wild-type human growth hormone. These results indicate that Tyr164 is required for the lactogenic activity of human growth hormone and that mutation to glutamic acid disrupts the lactogenic function of site 2.     

Studies of pituitary lactogenic hormone. Physicochemical characterization on procine prolactin.

Porcine pituitary lactogenic hormone has been examined by exclusion chromatography, analytical ultracentrifugation, free-boundary electrophoresis, solubility behavior, circular dichroism, and fluorescence spectra. The results have been compared with previous studies of ovine pituitary lactogenic hormone. These two hormones were found to resemble one another very closely by all criteria studied. While the homology between these lactogenic hormones and growth hormones is strongly reflected in the sharing of certain physicochemical properties, a number of distinct structural differences between these two closely related evolutionary lines are now becoming apparent.     

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.     

Prolactin Synthesis in Primates

THERE is compelling physiological evidence that prolactin and growth hormone in primates are separate proteins, but no conclusive chemical evidence has been obtained in support of this¹. Indeed the close similarity in the primary structure of human pituitary growth hormone (HGH) and ovine pituitary lactogenic hormone² has reinforced the view that perhaps, in the human, lactation and growth are controlled by a single pituitary protein. Histological and immunofluorescence studies using antiserum to ovine prolactin (AOP)³ have shown, however, that there is a substance in primate pituitaries which is immunochemically related to ovine prolactin (OP) and distinguishable from growth hormone.     

Theoretical analysis of the amino acid sequence of receptors for growth hormones and prolactins. Prediction of ligand-binding segments]

At present the growth hormone and prolactin receptors were cloned along with their variant forms from human, rat, mouse, rabbit, bovine and sheep tissues. The functional topography of receptors is practically unknown. Because of the high price and difficulty of protein's total mutagenesis, it is reasonable to carry on a theoretical analysis of structures of receptors to predict the most probable ligand-binding sites. We studied the primary structures of known prolactin and growth hormone receptors using theoretical methods proved to be powerful in earlier structure--activity relationship investigations. We analyzed the secondary structure, conservative positions, hydrophilicity profiles of the growth hormone and prolactin receptors, and used the original method based on information theory to predict the sites which are promising for mutagenesis or peptide synthesis as probable ligand-binding sites. Three segments corresponding to the main conservative, hydrophilic and rare sites were predicted to form the ligand-binding determinant.     

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.     

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.     

Interactions of antisense peptides with ovine prolactin

Two antisense peptides were synthesized to a sense peptide corresponding to amino acid residues 23-35 of ovine prolactin. Both of the antisense peptides formed a saturable complex with the sense peptide and ovine prolactin. The sense peptide inhibited the interaction of ovine prolactin with the antisense peptides. Both of the antisense peptides have a common core sequence VMNV which can bind to ovine prolactin. The lactogenic hormones, rat prolactin and human growth hormone, compete with the binding of ovine prolactin to an antisense peptide whereas a nonlactogen, ovine growth hormone, does not compete indicating a degree of specificity in the interaction.     

Analysis of the structure of prolactin terminal fragments as potential substrates of serine and proline-specific proteinases]

The biochemical mechanism of action of prolactin is unknown. This hormone enters the blood stream and binds to receptors predominantly in the monomeric form. A structural analysis of mammalian and piscine prolactin based on the present-day concepts of proteolytic processing of the hormone molecules in target tissues has been carried out. The experimental data suggest that prolactin molecules are protected from exopeptidase influence by their terminal cyclic peptides. The highly conservative proline-2 residues increase the resistance of the mammalian hormone N-terminal fragment to the effects of many aminopeptidases. Structurally the C-terminal cyclic peptides of prolactin, growth hormone and placental lactogen were shown to be homologous to peptides inhibiting trypsin-like proteinases. A structural analysis of the N-terminal domain of mammalian prolactin revealed the important role of Pro-2 and Pro-4 residues at positions adjacent with and inside the disulfide moiety. It is assumed that these proline residues and the cyclic structure are necessary for the manifestation of the inhibiting effect of the mammalian prolactin N-terminal dodecapeptide on proline-specific proteinases. It is assumed that proteolytic degradation of prolactin molecules in target tissues may induce the secretion of functionally active peptides.     

Reaction of the histidines of prolactin with ethoxyformic anhydride. A binding site modification.

The seven histidines of bovine prolactin were modified with ethoxyformic anhydride and two classes of reactivity were apparent: 5 histidines were in the more reactive class (k = 0.097 min-1) and 2 histidines were less reactive (k = 0.011 min-1). The activity of the modified prolactins was determined by measuring their ability to bind to prolactin receptors from rabbit mammary glands. This assay showed that prolactin was fully active when 0 to 5 histidines were modified. If all 7 residues were modified, the hormone was unable to bind to its receptor. Circular dichroism studies indicated no significant differences in conformation for prolactins which had 2 to 7 histidines modified. Modification of human growth hormone and human placental lactogen with ethoxyformic anhydride resulted in a loss of the ability of these lactogenic hormones to bind to the prolactin receptor. For all three hormones, essentially full activity was recovered when the modifying group was removed by treatment with hydroxylamine. Sequence comparisons indicate that only 2 of the 3 growth hormone histidines and 2 of 7 placental lactogen histidines were homologous with histidines in bovine prolactin and that, in each case, they correspond to His-27 and His-30 in bovine prolactin. It is postulated that these residues serve to identify a portion of the binding domain of bovine prolactin.     

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.     

Solubilization and characterization of a lactogenic receptor from human placental chorion membranes

Prolactin has a wide range of actions, including osmoregulation and the control of mammary gland development and lactation. These effects are mediated through a high-affinity cell surface receptor, which has been well characterized in a number of animal tissues. The molecular characteristics of the human receptor are unknown, however. The present studies were initiated, therefore, to determine the binding and molecular characteristics of the lactogenic receptor of human placental chorion membranes. Subcellular fractionation studies showed that the bulk of the receptor sedimented in the microsomal fraction at 45,000gav. Endogenous ligand was dissociated from the receptor with 3.5 M MgCl2 or 0.05 M acetate buffer (pH 4.8) with preservation of binding activity. The microsomal receptor bound human growth hormone (hGH), human prolactin (hPRL), ovine prolactin (oPRL), and human placental lactogen (hPL) but not non-primate growth hormones, indicating a narrow specificity for lactogenic hormones. The binding was only partially reversible in agreement with the known binding kinetics of animal lactogenic receptors. The receptor was solubilized with 45% yield from the microsomes using 16 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate (CHAPS) detergent-250 mM NaCl, and the binding activity was fully restored by a two-fold dilution in the binding reaction to reveal a KD of 0.8 nM for hGH and a binding capacity of 200 fmol of specifically bound hGH per mg of microsomal protein. Gel filtration chromatography indicated the minimum molecular weight of the ligand-receptor complex was approximately 60,000 daltons, and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of covalently cross-linked 125I-hGH-receptor complexes revealed a molecular size of 58,000 daltons. When account was taken of the contribution of the ligand, a molecular weight of 36,000 for the receptor's binding domain was obtained. These data indicate that the chorion lactogenic receptor has very similar binding and molecular characteristics to the lactogenic receptors from other mammalian species. Chorion membranes are thus a convenient source of material for the further purification and characterization of the human lactogenic receptor.     

Solution structure of human prolactin

We report the solution structure of human prolactin determined by NMR spectroscopy. Our result is a significant improvement over a previous structure in terms of number and distribution of distance restraints, regularity of secondary structure, and potential energy. More significantly, the structure is sufficiently different that it leads to different conclusions regarding the mechanism of receptor activation and initiation of signal transduction. Here, we compare the structure of unbound prolactin to structures of both the homologue ovine placental lactogen and growth hormone. The structures of unbound and receptor bound prolactin/placental lactogen are similar and no noteworthy structural changes occur upon receptor binding. The observation of enhanced binding at the second receptor site when the first site is occupied has been widely interpreted to indicate conformational change induced by binding the first receptor. However, our results indicate that this enhanced binding at the second site could be due to receptor-receptor interactions or some other free energy sources rather than conformational change in the hormone. Titration of human prolactin with the extracellular domain of the human prolactin receptor was followed by NMR, gel filtration and electrophoresis. Both binary and ternary hormone-receptor complexes are clearly detectable by gel filtration and electrophoresis. The binary complex is not observable by NMR, possibly due to a dynamic equilibrium in intermediate exchange within the complex. The ternary complex of one hormone molecule bound to two receptor molecules is on the contrary readily detectable by NMR. This is in stark contrast to the widely held view that the ternary prolactin-receptor complex is only transiently formed. Thus, our results lead to improved understanding of the prolactin-prolactin receptor interaction.     

Recombinant DNA in the study of protein hormones

The structures of some genes of pituitary, hypothalamic and thyroid protein hormones are reviewed. Cys-elements in the 5'-enhancer area of prolactin and growth hormone genes were identified by some authors. The possibility of biosynthesis of two variants of growth hormone and two different biologically active calcitonin related peptides are considered as results of alternative pro-mRNA splicing after expression of growth hormone and calcitonin genes. LH-RH and GH-RH gene structures are presented. They code the primary structures of several peptide hormones with different biological activities. These peptides are released after proteolytic processing of high molecular weight protein precursors. The structural and functional organization of the glucocorticoid receptor is discussed.     

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.     

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.