Turning the growth hormone receptor on: Evidence that hormone binding induces subunit rotation

Atomistic molecular dynamics simulations have been used to investigate the conformational changes associated with the binding of human growth hormone (hGH) to the extracellular domains (ECD) of the human growth hormone receptor (hGHR), thereby shedding light on the mechanism of activation. It is shown that the removal of hGH from the hormone‐bound receptor complex results in a counter‐clockwise rotation of the twosubunits relative to each other by 30°–64° (average 45° ± 14°), in close agreement in terms of both the magnitude and direction of the rotation with that proposed based on mutagenesis experiments. In addition to providing evidence to support a rotational activation mechanism, the simulations have enabled the nature of the interaction interfaces in both the cytokine‐bound and unliganded hGHR states to be analyzed in detail. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Human metapneumovirus induces a profile of lung cytokines distinct from that of respiratory syncytial virus

Lung cytokine and chemokine production by BALB/c mice infected with human metapneumovirus (hMPV) was compared to respiratory syncytial virus (RSV)-infected mice. hMPV infection induced lower levels of the inflammatory cytokines interleukin-1 (IL-1), IL-6 and tumor necrosis factor alpha but was a more potent inducer of granulocyte-macrophage colony-stimulating factor and triggered a more sustained production of the CXC chemokine KC compared to RSV. hMPV was a stronger inducer of both alpha interferon (IFN-alpha) and IFN-gamma responses than RSV. In regard to immunomodulatory cytokines, hMPV failed to induce detectable IL-10 or IL-12p70 but was a potent inducer of IL-12 p40 subunit. The implications for hMPV pathogenesis are discussed.     

Gonadotropin-releasing hormone receptor binding in bovine anterior pituitary

Synthetic gonadotropin-releasing hormone (GnRH) was monoiodinated at a high specific radioactivity with 125I. The iodinated hormone retained full biological activity as assessed by the release of luteinizing hormone in vitro from bovine anterior pituitary tissue slices. Specific binding of 125I-labeled gonadotropin-releasing hormone of high affinity and low capacity was obtained using dispersed bovine anterior pituitary cells. The binding had sigmoid characteristics, compatible with the presence of more than one binding site. The subcellular fraction responsible for binding was identified with the plasma membranes. However, significant binding also occurred in the secretory granules fraction. The plasma membranes were solubilized with sodium dodecyl sulfate. Using gonadotropin-releasing hormone covalently coupled to a solid phase, a protein was purified by an affinity technique from the solubilized plasma membrane preparation which possessed similar binding propperties as plasma membranes, both intact and solubilized. The protein migrated as a single component on polyacrylamide gel in sodium dodecyl sulfate and the estimated molecular weight was 60 000. The character of the gonadotropin-releasing hormone concentration dependence binding as well as association kinetics were multiphasic and suggested the presence of more than one binding site. When analyzed by the Hill plot, the Hill coefficient of all binding curves was always greater than one which is compatible with positive cooperativity. This was further supported by the dissociation studies where the dissociation rate was inversely proportionate to both the gonadotropin-releasing hormone concentration and the time interval during which the gonadotropin-releasing hormone-gonadotropin-releasing hormone receptor protein complex was formed. Using difference chromatography, aggregation of the purified gonadotropin-releasing hormone receptor protein was demonstrated to occur upon its exposure to gonadotropin-releasing hormone. The formed macromolecular complexes bound preferentially 125I-labeled gonadotropin-releasing hormone. It is concluded that a single receptor protein is responsible for gonadotropin-releasing hormone binding in the bovine anterior pituitary. It is a part of the plasma membranes. Its interaction with gonadotropin-releasing hormone provokes transitions of the protein into different allosteric forms and this may be related to the biological effect of gonadotropin-releasing hormone on gonadotropin secretion.     

Differences in hepatic growth hormone receptor binding during development of normal and dwarf chickens

The aim of this study was to compare the growth hormone (GH) receptor in liver microsomal fractions of normal chickens (Dw) and chickens carrying the dwarf gene (dw). Specific binding of GH to its hepatic receptor was significantly higher for Dw embryos from d 14 till d 20 of incubation than for dw embryos. The difference in binding was due to a decreased binding capacity but not affinity in the livers of the dwarf embryos. The same binding pattern was found in livers of adult chickens: lower binding was again caused by a lower number of GH receptors and at this stage the difference was even clearer than during embryonic development. Binding studies on livers of growing chicks demonstrated that binding was low for both genotypes, but a small though significant difference between them remained. The cause of this decrease in number of GH receptors in dwarf birds has yet to be determined but may be due to the primary action of the dwarf gene.     

Human pituitary growth hormone: solubility in ammonium sulfate solutions

The solubility of human pituitary growth hormone in ammonium sulfate solutions has been investigated and compared under similar conditions with that of three related proteins: ovine pituitary growth hormone, bovine pituitary growth hormone, and human chorionic somatomammotropin.     

Growth hormone receptor structure in adipocytes from normal and growth hormone-deficient rats

Using cross-linking techniques, we compared the properties of the growth hormone (GH) receptor in freshly isolated adipocytes from normal rats, from GH deficient rats, and in preincubated adipocytes from normal rats. Bound [125I]iodo-hGH was cross-linked to adipocytes with disuccinimidyl suberate, and membrane proteins labelled with [125I]iodo-hGH were visualized using sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. All of the adipocytes tested exhibited a prominent Mr = 134,000 band and additional less intense bands in the presence of reductant. No significant differences in the overall banding pattern of membrane proteins were evident in reducing or nonreducing gels, using adipocytes from rats made GH deficient by hypophysectomy or by treatment with antibodies against rat GH, or in fresh and preincubated cells from normal rats. Taken together with binding studies, these findings suggest that differences in the ability of GH to stimulate glucose oxidation in rat adipose tissue probably involve differences distal to the GH receptor.     

The N-terminal fragment of human parathyroid hormone receptor 1 constitutes a hormone binding domain and reveals a distinct disulfide pattern

The N-terminal extracellular parts of human G-protein coupled receptor class B, for example, receptors for secretin, glucagon, or parathyroid hormone, are involved in ligand binding. To obtain structural and functional information on the N-terminal receptor fragment of human parathyroid hormone receptor 1 (PTHR1), the truncated receptor was expressed in the cytosol of Escherichia coli in the form of inclusion bodies. Oxidative refolding of inclusion body material resulted in stable, soluble, monomeric protein. Ligand binding was proved by surface plasmon resonance spectroscopy and isothermal titration calorimetry. Refolded receptor fragment was able to bind parathyroid hormone with an apparent dissociation constant of 3-5 microM. Far-UV circular dichroism spectra showed that the refolded polypeptide contained approximately 25% alpha-helical and 23% beta-sheet secondary structures. Analysis of the disulfide bond pattern of the refolded receptor fragment revealed disulfide bonds between Cys170 and Cys131, Cys148 and Cys108, and Cys117 and Cys48. These results demonstrate that the extracellular N-terminal domain of the parathyroid hormone receptor (PTHR1) possesses a well-defined, stable conformation, which shows a significant ligand binding activity.     

Human fibroblasts from normal and malignant breast tissue grown in vitro show a distinct senescence profile and telomerase activity

The telomerase activity and the senescence profile of cultured breast fibroblasts from normal human interstitial and malignant stromal tissue were studied in comparison with their proliferation and differentiation pattern. Fibroblasts were grown either in the presence or absence of a conditioned medium (CM) obtained from cultures of the oestrogen receptor-positive breast cancer MCF-7 cell line. At different passages (from the 2nd up to the 48th), fibroblasts were examined for the telomerase activity by the Telomerase Repeats Amplification Protocol (TRAP) assay, for proliferation profile by Ki-67 antigen expression, and the myofibroblast or smooth muscle cell-like differentiation pattern by immunofluorescence with monoclonal antibodies specific for smooth muscle markers. Serial passages of fibroblasts from normal or tumour breast reveal that the relationship between the levels of telomerase activity and phenotypic/proliferation profile changes with cell subcultivation in a different manner in the two cell populations. The fibroblasts from normal tissue completed 12 passages in a CM-independent way prior to senescence whereas fibroblasts from tumour stroma senescence were attained after 48 passages. These cells showed a marked decrease of telomerase activity, growth rate and smooth muscle -actin expressing myofibroblasts after the 32nd passage. CM treatment of this fibroblast population induces a decline in the myofibroblast content, which precedes the changes in telomerase activity. Passaged fibroblasts from normal breast tissue can be converted to myofibroblasts upon CM treatment whereas those from tumour stroma were CM-insensitive. Taken together our data suggest that a heterogeneous fibroblast population with different life span is activated/recruited in the breast interstitium and poses the problem of a unique activation/recruitment of fibroblasts in neoplastic conditions.     

Human pituitary growth hormone: immunochemical investigations of biologically active fragments

Guinea pig antisera to human growth hormone were tested for their ability to recognize the two biologically active fragments of the hormone produced by human plasmin digestion and a synthetic active fragment. A precipitin line was obtained with native human growth hormone, plasmin-treated human growth hormone, and its NH₂-terminal fragment (residues 1–134). In the microcomplement-fixation and radioimmunoassay experiments, the NH₂-terminal plasmin fragment (residues 1–134) showed a greater immunoreactivity than the COOH-terminal plasmin fragment (residues 141–191). This, in turn, was more active than the synthetic fragment (residues 95–136).     

Characterization of human growth hormone from acromegalic pituitary tumors

Human growth hormone (HGH) was extracted from acromegalic pituitary tumors at pH 10.5 and precipitated with ammonium sulfate at 20-40% saturation. It was purified on a Sephadex G-100 column to yield monomeric HGH. The tumor-HGH was indistinguishable from the authentic one in polyacrylamide gel electrophoresis at pH 8.3 or in the presence of sodium dodecyl sulfate, high-performance liquid chromatography, radioimmunoassay, peptide map, amino acid composition and N-terminal partial amino acid sequence. The tumor-HGH is active in the tibia assay and bodyweight gain test in hypophysectomized rats with comparable potency to that of the authentic sample.     

Identification of nuclear factors that enhance binding of the thyroid hormone receptor to a thyroid hormone response element

Using a gel shift assay, we analyzed the binding of in vitro translated alpha- and beta-thyroid hormone (T3) receptors to a T3-response element (TRE) derived from the rat GH gene. No receptor-TRE complexes were observed when translated receptor alone was incubated with the TRE. However, addition of a nuclear extract from liver to the translational products resulted in the formation of two receptor-DNA complexes for both the alpha- and beta-receptors. These complexes were shown to contain translated receptor by comigration of 32P-labeled TRE and 35S-labeled receptor in the gel shift assay. A competition experiment demonstrated that formation of the complexes was sequence specific. Preincubation of the liver nuclear extract at 60 C abolished formation of both complexes indicating that receptor-TRE binding required a heat-labile nuclear factor. Phosphocellulose chromatography of the nuclear extract resulted in separation of the activities required for formation of the two complexes. Analysis of nuclear extracts from different tissues revealed that one complex formed in the presence of all extracts, whereas the second complex appeared predominantly with a nuclear extract from liver. Addition of T3 to the binding reaction had no effect on receptor-TRE complex formation. We suggest that nuclear factors interact with the T3 receptor to enhance hormone-independent binding to a TRE.