A Novel Dysfunctional Growth Hormone Variant (Ile179Met) Exhibits a Decreased Ability to Activate the Extracellular Signal-Regulated Kinase Pathway

Abstract The pituitary-expressed GH1 gene was screened for mutation in a group of 74 children with familial short stature. Two novel mutations were identified: an Ile179Met substitution and a −360A→G promoter variant. The Ile179Met variant was shown to exhibit a similar degree of resistance to proteolysis as wild-type GH, indicating that the introduction of Met does not cause significant misfolding. Secretion of Ile179Met GH from rat pituitary cells was also similar to that of wild type. Although receptor binding studies failed to show any difference in binding characteristics, molecular modeling studies suggested that the Ile179Met substitution might nevertheless perturb interactions between GH and the GH receptor loop containing the hotspot residue Trp169, thereby affecting signal transduction. The ability of the Ile179Met variant to activate a signal transducer and activator of transcription (STAT) 5-responsive luciferase reporter gene and induce phosphorylation of STAT 5 and ERK was therefore studied. In contrast to its ability to activate STAT 5 normally, activation of ERK by the Ile179Met variant was reduced to half that observed with wild type. Although differential effects on the activation of distinct signaling pathways by a mutant receptor agonist are unprecedented, these findings also suggest that the ERK pathway could play a role in mediating the action of GH.

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Decreased Expression of the GHRH Receptor Gene Due to a Mutation in a Pit-1 Binding Site

Molecular Endocrinology ◽

10.1210/mend.16.3.0785 ◽

2002 ◽

Vol 16 (3) ◽

pp. 450-458 ◽

Cited By ~ 36

Author(s):

Roberto Salvatori ◽

Xiaoguang Fan ◽

Primus E. Mullis ◽

Azeb Haile ◽

Michael A. Levine

Keyword(s):

Dna Binding ◽

Gh Deficiency ◽

Chinese Hamster Ovary Cells ◽

Luciferase Reporter ◽

Pituitary Cells ◽

Wild Type ◽

Binding Studies ◽

Ghrh Receptor ◽

Deficiency Type ◽

Mutant Promoter

Abstract A variety of mutations in the gene encoding the GHRH receptor (GHRHR) that are predicted to alter protein structure or function have been recently described in patients with isolated GH deficiency type IB. In the present report we describe a patient with isolated GH deficiency type IB who was heterozygous for two novel mutations in this gene: a missense mutation in codon 329 that replaces lysine with glutamic acid (K329E) and an A→C transversion (position −124) in one of the two sites of the promoter region that binds the pituitary-specific transcription factor Pit-1, which is required for GHRHR expression. Chinese hamster ovary cells that were transfected with a cDNA encoding the K329E GHRHR expressed the receptor but failed to show a cAMP response after treatment with GHRH, confirming the lack of functionality. To test the effect of the A→C mutation at position −124 of the promoter, we transfected rat GH3 pituitary cells, which express endogenous Pit-1, with plasmids in which the luciferase reporter gene was under the control of either the wild-type or the mutant promoter. GH3 cells expressing the mutant promoter showed significantly less luciferase activity than cells expressing the wild-type promoter. DNA-binding studies confirmed that the A→C base change markedly reduces DNA binding to the Pit-1 protein. These results demonstrate that mutations in the GHRHR are not limited to the coding sequence and that promoter mutations that impair Pit-1 binding can reduce expression of the GHRHR gene.

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circAkap17b acts as a miR-7 family molecular sponge to regulate FSH secretion in rat pituitary cells

Journal of Molecular Endocrinology ◽

10.1530/jme-20-0036 ◽

2020 ◽

Vol 65 (4) ◽

pp. 135-148

Author(s):

Chang-Jiang Wang ◽

Fei Gao ◽

Yi-Jie Huang ◽

Dong-Xu Han ◽

Yi Zheng ◽

...

Keyword(s):

Noncoding Rna ◽

Molecular Mechanisms ◽

Target Genes ◽

Luciferase Reporter ◽

Circular Rnas ◽

Pituitary Cells ◽

Rat Pituitary ◽

Rna Immunoprecipitation ◽

Rat Pituitary Cells

The pituitary gland functions as a prominent regulator of diverse physiologic processes by secreting multiple hormones. Circular RNAs (circRNAs) are an emerging novel type of endogenous noncoding RNA that have recently been recognized as powerful regulators participating in various biological processes. However, the physiological roles and molecular mechanisms of circRNAs in pituitary remain largely unclear. Herein, we concentrated on expounding the biological function and molecular mechanism of circRNA in rat pituitary. In this study, we identified a novel circRNA in pituitary tissue, circAkap17b, which was pituitary- and stage-specific. Then, we designed circAkap17b siRNA and constructed an overexpression plasmid to evaluate the effect of loss- and gain-of-circAkap17b function on FSH secretion. Interestingly, silencing circAkakp17b significantly inhibited FSH expression and secretion, while overexpression of circAkap17b enhanced FSH expression and secretion. Furthermore, dual luciferase reporter and RNA immunoprecipitation (RIP) assays confirmed that circAkap17b could serve as miR-7 sponge to regulate target genes. Additionally, miR-7b suppressed FSH expression and secretion by directly targeting Fshb through the dual luciferase reporter and RT-qPCR analysis. Additionally, rescue experiments showed that circAkap17b could regulate FSH secretion in pituitary cells through a circAkap17b-miR-7-Fshb axis. Collectively, we demonstrated that circAkap17b could act as a molecular sponge of miR-7 to upregulate expression of the target gene Fshb and facilitate FSH secretion. These findings provide evidence for a novel regulatory role of circRNAs in pituitary.

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SPECIFIC INTERACTION OF CORTICOSTEROIDS WITH BINDING SITES IN THE PLASMA MEMBRANES OF THE RAT ANTERIOR PITUITARY GLAND

Journal of Endocrinology ◽

10.1677/joe.0.0790215 ◽

1978 ◽

Vol 79 (2) ◽

pp. 215-222 ◽

Cited By ~ 69

Author(s):

B. KOCH ◽

B. LUTZ-BUCHER ◽

B. BRIAUD ◽

C. MIALHE

Keyword(s):

Binding Sites ◽

Plasma Membranes ◽

Gradient Centrifugation ◽

Specific Interaction ◽

Sucrose Density Gradient ◽

Pituitary Cells ◽

Sucrose Density Gradient Centrifugation ◽

Binding Characteristics ◽

Rat Pituitary ◽

Rat Pituitary Cells

The binding of glucocorticoids to a crude fraction of rat pituitary plasma membranes and to solubilized membrane proteins was measured. The binding characteristics were similar to those exhibited by transcortin: radioactive corticosterone was bound to a greater extent than radioactive dexamethasone and labelled corticosterone, but not labelled dexamethasone, was displaced by unlabelled corticosterone, deoxycorticosterone and progesterone. A Scatchard plot of the binding data revealed the presence of a binding material with a dissociation constant of about 3·2 nmol/l, which sedimented at 4S after sucrose density-gradient centrifugation. It was found that the number of binding sites was inversely related to the concentration of corticosterone in the circulation and was increased after long-term adrenalectomy. These data suggest that a material similar to transcortin is complexed to the plasma membrane of rat pituitary cells.

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