Designing small-molecule switches for protein-protein interactions.
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Mutations introduced into human growth hormone (hGH) (Thr175 --> Gly-hGH) and the extracellular domain of the hGH receptor (Trp104 --> Gly-hGHbp) created a cavity at the protein-protein interface that resulted in binding affinity being reduced by a factor of 10(6). A small library of indole analogs was screened for small molecules that bind the cavity created by the mutations and restore binding affinity. The ligand 5-chloro-2-trichloromethylimidazole was found to increase the affinity of the mutant hormone for its receptor more than 1000-fold. Cell proliferation and JAK2 phosphorylation assays showed that the mutant hGH activates growth hormone signaling in the presence of added ligand. This approach may allow other protein-protein and protein-nucleic acid interactions to be switched on or off by the addition or depletion of exogenous small molecules. (+info)
Activation of extracellular-regulated kinase pathways in ovarian granulosa cells by the novel growth factor type 1 follicle-stimulating hormone receptor. Role in hormone signaling and cell proliferation.
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Follicle-stimulating hormone (FSH) regulated growth and function of the ovarian follicle was previously thought to be mediated solely through activation of G(s)-coupled receptors. In this study, we show for the first time that this function is predominantly mediated through the alternatively spliced and novel growth factor type 1 receptor (oFSH-R3) that is also present in the ovary. Immortalized granulosa cells lacking endogenous FSH receptors, when transfected with either oFSH-R3 cDNA (JC-R3) or the G(s)-coupled oFSH-R1 (JC-R1), expressed the corresponding glycosylated receptor. In JC-R3 or JC-R1 cells labeled with bromodeoxyuridine or [(3)H]thymidine, FSH stimulated the cells to progress through S-phase and divide. The growth promoting effect of recombinant FSH in JC-R3 cells was preceded by the rapid activation of ERK1 and ERK2. This effect was hormone-specific and transient. In JC-R3 cells inhibitors like calphostin C, PD98059, Ag 18, or calcium chelators EGTA or 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/AM inhibited both mitogen-activated protein kinase activation and bromodeoxyuridine incorporation. FSH induced phosphorylation of the FSH-R3 receptor was blocked by pretreating cells with calphostin C. There was no cAMP induction by FSH in JC-R3 cells. The cAMP independent growth promoting effect of FSH is mediated by activation of Ca(2+) and mitogen-activated protein kinase-dependent pathways. Thus, alternative splicing of a G-protein coupled receptor creates the expression of a novel receptor motif that can mediate a widely recognized function of the glycoprotein hormone. (+info)
Characterisation of novel missense mutations in the GH receptor gene causing severe growth retardation.
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Two Swedish brothers, 2.5 and 4 years of age, were found to fulfil all the clinical and laboratory characteristics of Laron's syndrome. They were shown to have unique missense mutations in the GH receptor gene. Both of their parents were of normal height, but they both separately carried one of the identified gene alterations. A molecular model of the first receptor alteration suggests that a collapse in three-dimensional receptor structure most likely contributed to the GH insensitivity in these patients. (+info)
Growth hormone receptor abundance in tibial growth plates of uremic rats: GH/IGF-I treatment.
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BACKGROUND: Children with chronic renal failure (CRF) exhibit growth retardation and a disturbed growth hormone/insulin-like growth factor-I (GH/IGF-I) axis. Treatment of children with CRF with GH or GH/IGF-I can partially restore linear growth. The molecular basis for decreased longitudinal growth is not known but may involve an impaired action of GH. METHODS: We used the growth-retarded uremic rat model to determine the abundance and distribution of GH receptors (GHRs) in the tibial epiphyseal growth plate and the influence of GH, IGF-I, or combined GH/IGF-I treatment. Pair-fed rats were used as the control. RESULTS: While all treatment regimes increased body length and weight in both rat groups, only GH/IGF-I treatment increased the total growth plate width. This involved an increase in cell number in the hypertrophic zone, which could also be induced by IGF-I alone. Immunohistochemical analysis showed that uremic rats had decreased abundance of GHRs in the proliferative zone, and only GH/IGF-I therapy could overcome this decrease. These data thus suggest that growth retardation in uremic rats is, at least in part, due to a decrease in GHR abundance in chondrocytes of the proliferative zone of the tibial growth plate. This decreased GHR abundance can be overcome by combined GH/IGF-I therapy, thus enhancing generation and proliferation of hypertrophic zone chondrocytes and increasing growth-plate width. CONCLUSION: These studies point to a mechanism for the growth retardation seen in children with CRF, and suggest that combined GH/IGF-I treatment may provide more effective therapy for these patients than GH alone. (+info)
Recurrence of sellar and suprasellar tumors in children treated with hGH--relation to immunohistochemical study on GH receptor.
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PURPOSE: GH replacement therapy is required in the majority of children with GH deficiency after treatment of sellar and suprasellar tumors. Owing to the high cell proliferative ability of human GH (hGH), its influence on tumor recurrence has been debated. We retrospectively studied the immunohistochemical expression of the GH receptor in various tumor tissues, in order to investigate the relation between tumor recurrence and hGH replacement. METHODS: GH replacement therapy was performed in 25 patients (8 boys and 17 girls) after the treatment. Tumor recurrence was noted in 4 patients (craniopharyngioma: 2 patients, pilocytic astrocytoma and germinoma: 1 each). Immunohistochemical study of GH receptor in tumor tissue was carried out in those recurrent and recurrence-free cases, by using MAb 263 as a primary antibody. RESULTS: Two patients with recurrent craniopharyngioma were positive for MAb 263, but 1 recurrence-free patient was negative. Patients with pilocytic astrocytoma (recurrent and recurrence-free: 1 each) were all positive. Five patients with germinoma (1 with recurrence and 4 without recurrence) were all negative. CONCLUSION: In the patients with craniopharyngioma treated with GH, a positive immunohistochemical expression of GH receptor in tumor tissue may indicate a high probability of recurrence. In our cases, GH receptor was positive in astrocytomas and negative in germinomas, with or without recurrence. It is therefore speculated that each brain tumor may have its specificity in GH receptor expression. (+info)
Charged residues dominate a unique interlocking topography in the heterodimeric cytokine interleukin-12.
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Human interleukin-12 (IL-12, p70) is an early pro-inflammatory cytokine, comprising two disulfide-linked subunits, p35 and p40. We solved the crystal structures of monomeric human p40 at 2.5 A and the human p70 complex at 2.8 A resolution, which reveals that IL-12 is similar to class 1 cytokine-receptor complexes. They also include the first description of an N-terminal immunoglobulin-like domain, found on the p40 subunit. Several charged residues from p35 and p40 intercalate to form a unique interlocking topography, shown by mutagenesis to be critical for p70 formation. A central arginine residue from p35 projects into a deep pocket on p40, which may be an ideal target for a small molecule antagonist of IL-12 formation. (+info)
Decreased growth in angus steers with a short TG-microsatellite allele in the P1 promoter of the growth hormone receptor gene.
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A polymorphic TG-repeat microsatellite is located 90 base pairs upstream from a major transcription start site in the bovine growth hormone receptor gene. A shorter allele with 11 consecutive TG is common in Bos indicus cattle, whereas longer 16- to 20-TG-repeat alleles predominate in Bos taurus breeds. The purpose of this study was to compare growth and carcass traits between Angus steers that had two of the longer growth hormone receptor alleles with their half-siblings that had one short allele and one of the longer alleles. We genotyped 64 Angus sires with respect to the poly-TG microsatellite and identified six bulls that were heterozygous in that they had one short 11-TG allele and one of the longer alleles. We then grouped 125 steer progeny of these six heterozygous bulls according to their genotypes: only the longer 16- to 20-TG-repeat alleles were found in 73 steer progeny (long/long homozygotes), whereas a short 11-TG allele was paired with one of the longer alleles in 52 progeny (short/long heterozygotes). Contrasts for the long/long homozygotes vs the short/long heterozygotes were significant for weaning weight (17 +/- 4 kg; P < .001) and carcass weight (14 +/- 5 kg; P < .01). Approaching significance (P = .03) was the contrast for USDA marbling score (-.3 +/- .2). No significant differences (P > .05) were detected for contrasts in birth weight (.3 +/- .6 kg), longissimus muscle area (-.2 +/- 1 cm2), or carcass fat depth (-.01 +/- .07 cm). This polymorphism has potential for use in DNA marker-assisted selection programs. (+info)
Growth and the initiation of steroidogenesis in porcine follicles are associated with unique patterns of gene expression for individual componentsof the ovarian insulin-like growth factor system.
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Ovarian follicular growth and steroidogenesis are controlled by the interaction of insulin-like growth factors (IGFs) and gonadotropins. The objective was to determine the temporal and spatial relationships for gonadotropin receptor, steroidogenic enzyme, and IGF system gene expression during the development of preovulatory porcine follicles. Sows (n = 18) were weaned and follicles were monitored by transrectal ultrasonography. Ovaries were collected from sows when the mean diameter of the preovulatory follicular cohort was approximately 2, 4, 6, or 8 mm. mRNA were measured by in situ hybridization for individual follicles within the preovulatory cohort (3 to 5 follicles per sow). Patterns of gene expression detected by in situ hybridization were confirmed by RNase protection analyses of pooled RNA samples. The amount of LH receptor mRNA and steroidogenic enzyme mRNA (17alpha-hydroxylase and aromatase) increased as the mean diameter of the follicular cohort increased from 2 to 6 mm, but then decreased abruptly for 8-mm follicles. Estradiol concentrations in follicular fluid closely followed the expression patterns of steroidogenic enzymes and LH receptor mRNA. FSH receptor mRNA was present in cohorts of 2-mm follicles but declined in 4-mm follicles and was undetectable in 6- and 8-mm follicles. The expression of IGF-I and type I IGF receptor mRNA were similar for follicles of 2, 4, 6, and 8 mm. In contrast, IGF-II mRNA progressively increased in follicles collected from 2-, 4-, and 6-mm cohorts, and then decreased slightly at 8 mm. Type II IGF receptor mRNA was greatest in 8-mm follicles. IGF binding protein-2 (BP-2) mRNA decreased as follicles achieved progressively larger sizes during the preovulatory period (2 to 8 mm), whereas the IGFBP-4 mRNA remained relatively low for follicles in 2- to 6-mm cohorts but then increased markedly in 8-mm follicles. In summary, temporal and spatial patterns of gene expression for gonadotropin receptor, steroidogenic enzyme, and IGF system genes were characterized in preovulatory porcine follicles by using in situ hybridization and RNase protection analyses. The unique patterns of gene expression suggest interdependence among specific genes that may be essential for preovulatory follicular development. (+info)