Ternary complex between placental lactogen and the extracellular domain of the prolactin receptor

The structure of the ternary complex between ovine placental lactogen (oPL) and the extracellular domain (ECD) of the rat prolactin receptor (rPRLR) reveals that two rPRLR ECDs bind to opposite sides of oPL with pseudo two-fold symmetry. The two oPL receptor binding sites differ significantly in their topography and electrostatic character. These binding interfaces also involve different hydrogen bonding and hydrophobic packing patterns compared to the structurally related human growth hormone (hGH)-receptor complexes. Additionally, the receptor-receptor interactions are different from those of the hGH-receptor complex. The conformational adaptability of prolactin and growth hormone receptors is evidenced by the changes in local conformations of the receptor binding loops and more global changes induced by shifts in the angular relationships between the N- and C-terminal domains, which allow the receptor to bind to the two topographically distinct sites of oPL.     

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.     

The WSXWS motif in cytokine receptors is a molecular switch involved in receptor activation: insight from structures of the prolactin receptor

The prolactin receptor (PRLR) is activated by binding of prolactin in a 2:1 complex, but the activation mechanism is poorly understood. PRLR has?a conserved WSXWS motif generic to cytokine class I receptors. We have determined the nuclear magnetic resonance solution structure of the membrane proximal domain of the human PRLR and find that the tryptophans of the motif adopt a T-stack conformation in the unbound state. By contrast, in the hormone bound state, a Trp/Arg-ladder is formed. The conformational change is hormone-dependent and influences the receptor-receptor dimerization site 3. In the constitutively active, breast cancer-related receptor mutant PRLR(I146L), we observed a stabilization of the dimeric state and a change in the dynamics of the motif. Here we demonstrate a structural link between the WSXWS motif, hormone binding, and receptor dimerization and propose it?as?a general mechanism for class 1 receptor activation.     

The purification and characterization of rabbit placental lactogen.

Rabbit placental lactogen, a polypeptide hormone functionally related to the growth hormone/prolactin family, was isolated from placenta by (NH4)2SO4 precipitation, gel filtration and ion-exchange chromatography on DEAE-and CM-cellulose. The hormone was purified to more than 90% homogeneity, as determined by end-group analysis. On disc gel electrophoresis at pH9.0 it migrates as a pair of closely spaced bands with mobilities of 0.489 (minor band) and 0.511 (major band), and its isoelectric point is 6.1. Its mol.wt. is 20600, as determined by sedimentation--equilibrium centrifugation, and 24200, as estimated by gel electrophoresis in sodium dodecyl sulphate. Its amino acid composition resembles that of rabbit growth hormone and rat prolactin, except for a lower glutamic acid and leucine content. Like the prolactins, rabbit placental lactogen has two tryptophan and six cysteine residues, and its N-terminus, valine, is identical with that for human placental lactogen. By radioimmunoassay, it does not cross-react with antisera to either rat growth hormone or rat prolactin; in addition, it does not cross-react with antisera to bovine placental lactogen by double immunodiffusion. The similarity of the biochemical characteristics of rabbit placental lactogen to the other non-primate placental lactogens lends further support to the hypothesis that these molecules occupy a more central position in the growth hormone/prolactin "tree" than do their primate counterparts.     

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.     

The tertiary structure and backbone dynamics of human prolactin

Human prolactin is a 199-residue (23 kDa) protein closely related to growth hormone and placental lactogen with properties and functions resembling both a hormone and a cytokine. As a traditional hormone, prolactin is produced by lactotrophic cells in the pituitary and secreted into the bloodstream where it acts distally to regulate reproduction and promote lactation. Pituitary cells store prolactin in secretory granules organized around large prolactin aggregates, which are produced within the trans layer of the Golgi complex. Extrapituitary prolactin is synthesized by a wide variety of cells but is not stored in secretory granules. Extrapituitary prolactin displays immunomodulatory activities and acts as a growth factor for cancers of the breast, prostate and tissues of the female reproductive system. We have determined the tertiary structure of human prolactin using three-dimensional (3D) and four-dimensional (4D) heteronuclear NMR spectroscopy. As expected, prolactin adopts an "up-up-down-down" four-helical bundle topology and resembles other members of the family of hematopoietic cytokines. Prolactin displays three discrete structural differences from growth hormone: (1) a structured N-terminal loop in contact with the first helix, (2) a missing mini-helix in the loop between the first and second helices, and (3) a shorter loop between the second and third helices lacking the perpendicular mini-helix observed in growth hormone. Residues necessary for functional binding to the prolactin receptor are clustered on the prolactin surface in a position similar to growth hormone. The backbone dynamics of prolactin were investigated by analysis of 15N NMR relaxation phenomena and demonstrated a rigid four-helical bundle with relatively mobile interconnecting loops. Comparison of global macromolecular tumbling at 0.1mM and 1.0mM prolactin revealed reversible oligomerization, which was correlated to dynamic light scattering experiments. The existence of a reversible oligomerization reaction in solution provides insight into previous results describing the in vitro and in vivo aggregation properties of human prolactin.     

A receptor site for prolactin in lactating mouse mammary tissues.

Prolactin iodinated by lactoperoxidase method showed immunologically, electrophoretically and biolo9gically similar properties to native prolactin and possessed enough specific radioactivity for receptor studies. 1251-prolactin was incubated with mouse mammary tissues at 8 days of lactation. Both binding and release of 1251-prolactin depended on incubation time and temperature and were maximal at 37 degrees C. Michaelis constant was estimated to be 1.4 X 10(-9) M from Lineweaver-Burk plot and to be 1.2 X 10(-9) M from id-value of the dose-response curve for displacement with native prolactin. Total number of binding sites for prolactin was 1.38 X 10(-15) mole per mg weight of tissue. Ovine prolactin, human growth hormone and human placental lactogen complete with 1251-prolactin and dose-response curves for these three hormones were all parallel. These results suggest the existence of a specific receptor site with high affinity for prolactin in lactating mouse mammary glands.     

Crystal structure and site 1 binding energetics of human placental lactogen

In primates, placental lactogen (PL) is a pituitary hormone with fundamental roles during pregnancy involving fetal growth, metabolism, and stimulating lactation in the mother. Human placental lactogen (hPL) is highly conserved with human growth hormone (hGH) and both hormones bind to the hPRLR extracellular domain (ECD), the first step in receptor homodimerization, in a Zn2+-dependent manner. A modified surface plasmon resonance method was developed to measure the kinetics for hPL and hGH binding to the hPRLR ECD, with and without Zn2+ and showed that hPL has about a tenfold higher affinity for the hPRLR ECD1 than hGH. The crystal structure of the free state of hPL has been determined to 2.0 A resolution showing the molecule possesses an overall structure similar to other long chain four-helix bundle cytokines. Comparison of the free hPL structure with the 1:1 complex structure of hGH bound to the hPRLR ECD1 suggests that two surface loops undergo conformational changes >10 A upon binding. An 18 residue Ala-scan was used to characterize the binding energy epitope for the site 1 interface of hPL. Individual alanine substitutions at five positions reduced binding affinity by a DeltaDeltaG > or = 3 kcal mol(-1). A comparison of the hPL site 1 epitope with that previously determined for hGH indicates contributions of individual residues track reasonably well between hPL and hGH. In particular, residues involved in the zinc-binding site and Lys172 constitute the principal binding determinants for both hormones. However, several residues that are identical between hPL and hGH contribute quite differently to the binding of the hPRLR ECD1. Additionally, the overall magnitudes of the DeltaDeltaG changes observed from the Ala-scan of hPL were markedly larger than those determined in the comparative scan of hGH to the hPRLR ECD1. The structural and biophysical data presented here show that subtle changes in the structural context of an interaction can lead to significantly different effects at the individual residue level.     

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.     

Biological activity of bovine placental lactogen in 3T3-F442A preadipocytes is mediated through a somatogenic receptor.

Bovine placental lactogen (bPL) exhibited antimitogenic differentiation-promoting biological activity in 3T3-F442A preadipocytes. Competitive binding studies and affinity labelling revealed bPL activity to be mediated through a somatogenic type of receptor that recognizes human growth hormone (hGH) and bovine GH, but not ovine prolactin or human PL. The bioactivity of bPL was sixfold lower than that of hGH despite that bPL is binding to the somatogenic receptors with fivefold higher affinity. This discrepancy may result from the relatively low ability of bPL to induce post-receptoral effects such as receptor dimerization.     

Isolation and identification of a cDNA clone of rat placental lactogen II

The developing rat placenta expresses two placental lactogens at different stages of pregnancy: rat placental lactogen I from Days 11 to 13 of pregnancy and rat placental lactogen II (rPLII) from Day 12 to term. In this paper, we describe cDNA clones for rPLII, which have been isolated from a Day 18 rat placental cDNA library. The rPLII clones hybrid-select a mRNA which translates in vitro to a protein of 25,000 daltons. This protein is processed by dog pancreatic microsomes to a 22,000-dalton form, identical in size to rPLII isolated from pregnant rat serum. Both forms are precipitated by an anti-rPLII antiserum and an anti-ovine prolactin antiserum. The mRNA for rPLII is first expressed in Day 12 placenta and reaches a maximum at about Day 18 of pregnancy, in parallel with the appearance of the hormone in serum. Sequencing of the cDNA shows that, unlike human placental lactogen which is 85% homologous to human growth hormone at the amino acid level, rPLII is much more closely related to the prolactins. Thus, rPLII is 52% homologous to rat prolactin at the amino acid level, but only 34% related to rat growth hormone. This is the second placental lactogen to be fully characterized, and in the rat this hormone appears to have evolved by a route quite different from that which produced placental lactogen in humans.     

Properties of a prolactin receptor from the rabbit mammary gland

Receptors for human, simian, ovine, bovine and murine prolactin, human growth hormone and human placental lactogen have been identified in plasma-membrane-containing subcellular particles isolated from rabbit mammary glands. The association and dissociation of (125)I-labelled prolactin are time- and temperature-dependent processes, both being maximal at 37 degrees C. (125)I-labelled prolactin prepared by the enzymic iodination procedure with lactoperoxidase binds better to receptors than does the preparation obtained by using chloramine-t as the oxidizing agent. The binding of (125)I-labelled prolactin to receptors is strongly influenced by pH and ionic composition but not by many low-molecular-weight compounds tested, e.g. steroids, nucleotides and several drugs. Receptor activity is sensitive to trypsin and phospholipase C digestion, suggesting that protein and phospholipid moieties are essential for the binding of (125)I-labelled prolactin. The binding of (125)I-labelled prolactin to receptors is a saturable and reversible process. Scatchard and Lineweaver-Burk analyses suggest that (125)I-labelled prolactin has a high affinity for its receptor. Binding of (125)I-labelled prolactin to receptors does not result in the destruction of the hormone. Considerable prolactin-binding activity is also observed in subcellular fractions isolated from the adrenal gland, liver, ovary and kidney of the pregnant rabbit, a finding that is consistent with other reported actions of prolactin in these organs.     

Crystal structure of a prolactin receptor antagonist bound to the extracellular domain of the prolactin receptor

The crystal structure of the complex between an N-terminally truncated G129R human prolactin (PRL) variant and the extracellular domain of the human prolactin receptor (PRLR) was determined at 2.5A resolution by x-ray crystallography. This structure represents the first experimental structure reported for a PRL variant bound to its cognate receptor. The binding of PRL variants to the PRLR extracellular domain was furthermore characterized by the solution state techniques, hydrogen exchange mass spectrometry, and NMR spectroscopy. Compared with the binding interface derived from mutagenesis studies, the structural data imply that the definition of PRL binding site 1 should be extended to include residues situated in the N-terminal part of loop 1 and in the C terminus. Comparison of the structure of the receptor-bound PRL variant with the structure reported for the unbound form of a similar analogue ( Jomain, J. B., Tallet, E., Broutin, I., Hoos, S., van Agthoven, J., Ducruix, A., Kelly, P. A., Kragelund, B. B., England, P., and Goffin, V. (2007) J. Biol. Chem. 282, 33118-33131 ) demonstrates that receptor-induced changes in the backbone of the four-helix bundle are subtle, whereas large scale rearrangements and structuring occur in the flexible N-terminal part of loop 1. Hydrogen exchange mass spectrometry data imply that the dynamics of the four-helix bundle in solution generally become stabilized upon receptor interaction at binding site 1.     

Pre-parturitional changes in serum prolactin, placental lactogen, growth hormone, progesterone, and corticosterone in the C3H/HeN mouse

Pre-parturitional changes in serum prolactin, placental lactogen, growth hormone, progesterone, and corticosterone in the C3H/HeN mouse are described. Serum prolactin concentrations display an apparent biphasic pre-parturitional increase. Both serum placental lactogen and growth hormone concentrations are elevated during the second half of pregnancy. Serum placental lactogen concentrations remain elevated until parturition, whereas serum growth hormone concentrations decline on the last two days of pregnancy. Serum progesterone and corticosterone concentrations are elevated during the latter half of pregnancy and decline on the day preceding parturition.     

Glucocorticoid modulation of prolactin receptors on mammary cells of lactating mice.

Prolactin receptors were monitored by measuring 125I-labeled prolactin binding to collagenase-dissociated mammary epithelial cells of lactating BALB/c mice. Specific receptors for iodine-labeled prolactin with an apparent dissociation constant (Kd) of 0.99 . 10(-9) M were present on the dissociated mammary cells. The binding was inhibited by ovine prolactin, human growth hormone and human placental lactogen but not by follicle stimulating hormone, luteinizing hormone, thyroid stimulating hormone, bovine growth hormone or insulin. Adrenal ablation of nursing mothers caused a reduction of the number of prolactin receptors and this effect was preventable by hydrocortisone therapy. Hydrocortisone injections to mothers 3 days after adrenalectomy also induced a replenishment of the prolactin receptors on the mammary cells. Injections of progesterone failed to sustain the high level of mammary cell prolactin receptors in adrenalectomized animals. Stimultaneous injections of hydrocortisone and progesterone to animals 3 days after adrenalectomy caused a partial suppression of the stimulatory action of hydrocortisone alone. The results suggest that hydrocortisone can exert a modulatory influence on mammary cell prolactin receptors in non-hypophysectomized post-partum mice without altering the dissociation constant (Kd) of the receptors.     

Conformational changes in the active site loops of dihydrofolate reductase during the catalytic cycle

Escherichia coli dihydrofolate reductase (DHFR) has several flexible loops surrounding the active site that play a functional role in substrate and cofactor binding and in catalysis. We have used heteronuclear NMR methods to probe the loop conformations in solution in complexes of DHFR formed during the catalytic cycle. To facilitate the NMR analysis, the enzyme was labeled selectively with [(15)N]alanine. The 13 alanine resonances provide a fingerprint of the protein structure and report on the active site loop conformations and binding of substrate, product, and cofactor. Spectra were recorded for binary and ternary complexes of wild-type DHFR bound to the substrate dihydrofolate (DHF), the product tetrahydrofolate (THF), the pseudosubstrate folate, reduced and oxidized NADPH cofactor, and the inactive cofactor analogue 5,6-dihydroNADPH. The data show that DHFR exists in solution in two dominant conformational states, with the active site loops adopting conformations that closely approximate the occluded or closed conformations identified in earlier X-ray crystallographic analyses. A minor population of a third conformer of unknown structure was observed for the apoenzyme and for the disordered binary complex with 5,6-dihydroNADPH. The reactive Michaelis complex, with both DHF and NADPH bound to the enzyme, could not be studied directly but was modeled by the ternary folate:NADP(+) and dihydrofolate:NADP(+) complexes. From the NMR data, we are able to characterize the active site loop conformation and the occupancy of the substrate and cofactor binding sites in all intermediates formed in the extended catalytic cycle. In the dominant kinetic pathway under steady-state conditions, only the holoenzyme (the binary NADPH complex) and the Michaelis complex adopt the closed loop conformation, and all product complexes are occluded. The catalytic cycle thus involves obligatory conformational transitions between the closed and occluded states. Parallel studies on the catalytically impaired G121V mutant DHFR show that formation of the closed state, in which the nicotinamide ring of the cofactor is inserted into the active site, is energetically disfavored. The G121V mutation, at a position distant from the active site, interferes with coupled loop movements and appears to impair catalysis by destabilizing the closed Michaelis complex and introducing an extra step into the kinetic pathway.     

Human growth hormone-variant demonstrates a receptor binding profile distinct from that of normal pituitary growth hormone

We have recently established that the human growth hormone-variant (hGH-V) gene is functional in vivo by documenting its expression in the placenta. We have subsequently generated transformed murine cell lines stably expressing the genes for normal pituitary growth hormone (hGH-N), hGH-V, and each of two chimeric genes generated by exon 3 exchanges, hGH-NV3 and hGH-VN3. In the present study, we utilize these cell lines as sources of hormone to characterize and compare the receptor binding profiles of hGH-N with hGH-V. hGH-V was found to displace 125I-ovine prolactin bound to rat liver microsomes (lactogen binding) and to displace 125I-hGH bound to rabbit liver microsomes (somatogen binding). Therefore, hGH-V would be predicted to display both somatogenic and lactogenic bioactivity, a dual specificity previously thought to be unique to hGH-N. The concentrations of hormone necessary to displace 50% (IC50) of the 125I-hGH from somatogen receptors and 125I-ovine prolactin from lactogen receptors was expressed as a ratio, IC50 somatogen: IC50 lactogen, for each hormone tested. A 7.4-fold difference in this ratio was observed for hGH-N compared to hGH-V, suggesting significantly greater selectivity by hGH-V in binding to the somatogen receptor. The intermediate binding ratios of the hGH-NV3 and hGH-VN3 chimeric proteins confirmed the distinct receptor binding profiles of the two parent hormones and served to identify three amino acids of potential importance in defining their respective receptor binding specificities.     

Dimeric ("big") human placental lactogen. Immunological and biological activity.

Dimeric ("big") human placental lactogen has been isolated in near homogeneous form from placental tissue. It consists of a disulfide-linked (stable) form and a noncovalently associated (unstable) form of the native hormone. The two forms were separated by exposure to denaturing conditions and resolution by gel exclusion chromatography. Both forms retained immunological activity, ability to bind mammary membranes, and ability to induce mammary N-acetyllactosamine synthetase in vitro. On a molar basis, stable dimeric placental lactogen was more active than placental lactogen in the radioimmunoassay indicating that the immunological determinants on both monomeric units could bind to antibody. On a molar basis, stable dimeric placental lactogen was equally active with monomeric placental lactogen in competing for mammary gland membrane binding sites, indicating that only one active site in the molecule could interact with the membrane at a time. Stable dimeric placental lactogen was also active in an in vitro bioassay using the induction of N-acetyllactosamine synthetase. It is concluded that dimer formation does not alter the biologically active portion of the placental lactogen molecule. Since the carboxyl-terminal region (residues 182-191) is involved in the interchain disulfide bonds of dimeric placental lactogen, this portion of the molecule is probably not necessary for its biological activity.