Differential effects of follicle-stimulating hormone glycoforms on the transcriptome profile of cultured rat granulosa cells as disclosed by RNA-seq.

It has been documented that variations in glycosylation on glycoprotein hormones, confer distinctly different biological features to the corresponding glycoforms when multiple in vitro biochemical readings are analyzed. We here applied next generation RNA sequencing to explore changes in the transcriptome of rat granulosa cells exposed for 0, 6, and 12 h to 100 ng/ml of four highly purified follicle-stimulating hormone (FSH) glycoforms, each exhibiting different glycosylation patterns: a. human pituitary FSH18/21 (hypo-glycosylated); b.

Estrogen regulated transcription of the non-estrogen-regulated hamster uteroglobin gene in MCF-7 cells.

To study the estrogen regulated transcription of the uteroglobin (UG) gene, the founding member of the secretoglobin family widely expressed in many different mammalian species, we re-created functional estrogen response elements (EREs) in the UG gene promoter from a species where UG expression is not regulated by estrogens: the hamster Mesocricetus auratus (Ma), to ascertain if the lack of functional EREs is the real cause of its estrogen insensitivity. Functional EREs in the hamster promoter, including the consensus ERE (cERE), failed to respond to an appropriate estrogen stimulus compared with its estrogen regulated ortholog from the brown hare Lepus capensis (Lc). As the nucleotide sequence is the only difference between genetic constructs from these two species, we suspected that the UG promoter from the hamster probably contains cis-acting genetic elements that negatively impairs the estrogen-regulated transcription mediated by the functional ERE.

Differential effects of follicle-stimulating hormone glycoforms on the transcriptome profile of cultured rat granulosa cells as disclosed by RNA-seq.

It has been documented that variations in glycosylation on glycoprotein hormones, confer distinctly different biological features to the corresponding glycoforms when multiple biochemical readings are analyzed. We here applied next generation RNA sequencing to explore changes in the transcriptome of rat granulosa cells exposed for 0, 6, and 12 h to 100 ng/ml of four highly purified follicle-stimulating hormone (FSH) glycoforms, each exhibiting different glycosylation patterns: human pituitary FSH and equine FSH (FSH) (hypo-glycosylated), and human FSH and chinese-hamster ovary cell-derived human recombinant FSH (FSH) (fully-glycosylated). Total RNA from triplicate incubations was prepared from FSH glycoform-exposed cultured granulosa cells obtained from DES-pretreated immature female rats, and RNA libraries were sequenced in a HighSeq 2500 sequencer (2 × 125 bp paired-end format, 10-15 × 10 reads/sample).

Targeting trafficking as a therapeutic avenue for misfolded GPCRs leading to endocrine diseases.

G protein-coupled receptors (GPCRs) are plasma membrane proteins associated with an array of functions. Mutations in these receptors lead to a number of genetic diseases, including diseases involving the endocrine system. A particular subset of loss-of-function mutant GPCRs are misfolded receptors unable to traffic to their site of function ( the cell surface plasma membrane).

Misfolded G Protein-Coupled Receptors and Endocrine Disease. Molecular Mechanisms and Therapeutic Prospects.

Misfolding of G protein-coupled receptors (GPCRs) caused by mutations frequently leads to disease due to intracellular trapping of the conformationally abnormal receptor. Several endocrine diseases due to inactivating mutations in GPCRs have been described, including X-linked nephrogenic diabetes insipidus, thyroid disorders, familial hypocalciuric hypercalcemia, obesity, familial glucocorticoid deficiency [melanocortin-2 receptor, MC2R (also known as adrenocorticotropin receptor, ACTHR), and reproductive disorders. In these mutant receptors, misfolding leads to endoplasmic reticulum retention, increased intracellular degradation, and deficient trafficking of the abnormal receptor to the cell surface plasma membrane, causing inability of the receptor to interact with agonists and trigger intracellular signaling.

Placentas associated with female neonates from pregnancies complicated by urinary tract infections have higher cAMP content and cytokines expression than males.

Problem: The cAMP pathway is involved in important biological processes including immune regulation and hormone signaling. At the feto-maternal unit, cAMP participates in placental function/physiology and the establishment of immunoendocrine networks. Low cAMP in male fetuses cord blood has been linked to poorer perinatal outcomes; however, cAMP placental content and its relationship with immune factors and fetal sex in an infectious condition have not been investigated.

Structure-Function Relationships of the Follicle-Stimulating Hormone Receptor.

The follicle-stimulating hormone receptor (FSHR) plays a crucial role in reproduction. This structurally complex receptor is a member of the G-protein coupled receptor (GPCR) superfamily of membrane receptors. As with the other structurally similar glycoprotein hormone receptors (the thyroid-stimulating hormone and luteinizing hormone-chorionic gonadotropin hormone receptors), the FSHR is characterized by an extensive extracellular domain, where binding to FSH occurs, linked to the signal specificity subdomain or hinge region.

Molecular dynamics simulation of the follicle-stimulating hormone receptor. Understanding the conformational dynamics of receptor variants at positions N680 and D408 from in silico analysis.

Follicle-stimulating hormone receptor (FSHR) is a G-protein coupled receptor (GPCR) and a prototype of the glycoprotein hormone receptors subfamily of GPCRs. Structural data of the FSHR ectodomain in complex with follicle-stimulating hormone suggests a "pull and lift" activation mechanism that triggers a conformational change on the seven α-helix transmembrane domain (TMD). To analyze the conformational changes of the FSHR TMD resulting from sequence variants associated with reproductive impairment in humans, we set up a computational approach combining helix modeling and molecular simulation methods to generate conformational ensembles of the receptor at room (300 K) and physiological (310 K) temperatures.

Frequency of the T307A, N680S, and -29G>A single-nucleotide polymorphisms in the follicle-stimulating hormone receptor in Mexican subjects of Hispanic ancestry.

Background: FSHR SNPs may influence the ovarian sensitivity to endogenous and exogenous FSH stimulation. Given the paucity of data on the FSHR c.919A > G, c.

Intracellular Trafficking of Gonadotropin Receptors in Health and Disease.

Gonadotropin receptors belong to the highly conserved subfamily of the G protein-coupled receptor (GPCR) superfamily, the so-called Rhodopsin-like family (class A), which is the largest class of GPCRs and currently a major drug target. Both the follicle-stimulating hormone receptor (FSHR) and the luteinizing hormone/chorionic gonadotropin hormone receptor (LHCGR) are mainly located in the gonads where they play key functions associated to essential reproductive functions. As any other protein, gonadotropin receptors must be properly folded into a mature tertiary conformation compatible with quaternary assembly and endoplasmic reticulum export to the cell surface plasma membrane.

The Follitropin Receptor: Matching Structure and Function.

Follitropin, or follicle-stimulating hormone (FSH) receptor (FSHR), is a G protein-coupled receptor belonging to the glycoprotein hormone receptor family that plays an essential role in reproduction. Although its primary location is the gonad, the FSHR has also been reported in extragonadal tissues including bone, placenta, endometrium, liver, and blood vessels from a number of malignant tumors. The recently resolved crystal structure of FSH bound to the entire FSHR ectodomain has been instrumental in more clearly defining the role of this domain in ligand binding and receptor activation.

Role of Cysteine Residues in the Carboxyl-Terminus of the Follicle-Stimulating Hormone Receptor in Intracellular Traffic and Postendocytic Processing.

Posttranslational modifications occurring during the biosynthesis of G protein-coupled receptors include glycosylation and palmitoylation at conserved cysteine residues located in the carboxyl-terminus of the receptor. In a number of these receptors, these modifications play an important role in receptor function and particularly, in intracellular trafficking. In the present study, the three cysteine residues present in the carboxyl-terminus of the human FSHR were replaced with glycine by site-directed mutagenesis.

Pharmacoperones: targeting therapeutics toward diseases caused by protein misfolding.

Pharmacoperones are hydrophobic molecule drugs that enter cells and serve as a molecular framework to cause misfolded mutant proteins to fold properly and adopt a stable conformation compatible with proper intracellular trafficking. Pharmacoperones have successfully been used experimentally to rescue function of some misfolded proteins (enzymes, receptors, channels) that lead to disease. Identification of pharmacoperones by high-throughput screens of drug libraries will likely provide new molecules that may be potentially useful to treat diseases caused by protein misfolding.

Genistein stimulates fatty acid oxidation in a leptin receptor-independent manner through the JAK2-mediated phosphorylation and activation of AMPK in skeletal muscle.

Obesity is a public health problem that contributes to the development of insulin resistance, which is associated with an excessive accumulation of lipids in skeletal muscle tissue. There is evidence that soy protein can decrease the ectopic accumulation of lipids and improves insulin sensitivity; however, it is unknown whether soy isoflavones, particularly genistein, can stimulate fatty acid oxidation in the skeletal muscle. Thus, we studied the mechanism by which genistein stimulates fatty acid oxidation in the skeletal muscle.

Mutations in G protein-coupled receptors that impact receptor trafficking and reproductive function.

G protein coupled receptors (GPCRs) are a large superfamily of integral cell surface plasma membrane proteins that play key roles in transducing extracellular signals, including sensory stimuli, hormones, neurotransmitters, or paracrine factors into the intracellular environment through the activation of one or more heterotrimeric G proteins. Structural alterations provoked by mutations or variations in the genes coding for GPCRs may lead to misfolding, altered plasma membrane expression of the receptor protein and frequently to disease. A number of GPCRs regulate reproductive function at different levels; these receptors include the gonadotropin-releasing hormone receptor (GnRHR) and the gonadotropin receptors (follicle-stimulating hormone receptor and luteinizing hormone receptor), which regulate the function of the pituitary-gonadal axis.

Normal testicular function without detectable follicle-stimulating hormone. A novel mutation in the follicle-stimulating hormone receptor gene leading to apparent constitutive activity and impaired agonist-induced desensitization and internalization.

Activating mutations in the follicle-stimulating hormone (FSH) receptor (FSHR) gene are rarely detected due to the absence of a clearly defined phenotype, particularly in men. We here report the biochemical features of a novel mutation in the first extracellular loop of the FSHR. The mutation (N431I) was detected in an asymptomatic man exhibiting normal spermatogenesis, suppressed serum FSH, and normal or elevated levels of biochemical markers of FSH action.

Cloning the uteroglobin gene promoter from the relic volcano rabbit (Romerolagus diazi) reveals an ancient estrogen-response element.

To gain further insight on the estrogen-dependent transcriptional regulation of the uteroglobin (UG) gene, we cloned the 5'-flanking region of the UG gene from the phylogenetically ancient volcano rabbit (Romerolagus diazi; Rd). The cloned region spans 812 base pairs (bp; -812/-1) and contains a noncanonical TATA box (TACA). The translation start site is 48 bp downstream from the putative transcription initiation site (AGA), and is preceded by a consensus Kozak box.

Biochemical mechanism of pathogenesis of human gonadotropin-releasing hormone receptor mutants Thr104Ile and Tyr108Cys associated with familial hypogonadotropic hypogonadism.

The pathogenic mechanisms whereby the Thr104Ile and Tyr108Cys mutations in the gonadotropin-releasing hormone receptor (GnRHR) gene cause hypogonadotropic hypogonadism in humans are unknown. Transient expression of Thr104Ile and Tyr108Cys mutants in COS-7 cells revealed that both GnRHR mutants neither bind nor respond to agonist. Removal of Lys191 rescued function of both mutants, while addition of a carboxyl-terminal targeting sequence only rescued function of the Thr104Ile mutant.

Dominant negative effects of human follicle-stimulating hormone receptor expression-deficient mutants on wild-type receptor cell surface expression. Rescue of oligomerization-dependent defective receptor expression by using cognate decoys.

Current evidence indicates that G protein-coupled receptors form dimers that may affect biogenesis and membrane targeting of the complexed receptors. We here analyzed whether expression-deficient follicle-stimulating hormone receptor (FSHR) mutants exert dominant negative actions on wild-type FSHR cell surface membrane expression. Co-transfection of constant amounts of wild-type receptor cDNA and increasing quantities of mutant (R556A or R618A) FSHR cDNAs progressively decreased agonist-stimulated cAMP accumulation, [(125)I]-FSH binding, and plasma membrane expression of the mature wild-type FSHR species.

Multiple facets of follicle-stimulating hormone receptor function.

Follicle-stimulating hormone (FSH) is a glycoprotein hormone produced by the anterior pituitary gland. This gonadotropin plays an essential role in reproduction. Its receptor (FSHR) belongs to the superfamily of G protein-coupled receptors (GPCR), specifically the family of rhodopsin-like receptors.

Functional and structural roles of conserved cysteine residues in the carboxyl-terminal domain of the follicle-stimulating hormone receptor in human embryonic kidney 293 cells.

The carboxyl-terminal segment of G protein-coupled receptors has one or more conserved cysteine residues that are potential sites for palmitoylation. This posttranslational modification contributes to membrane association, internalization, and membrane targeting of proteins. In contrast to other members of the glycoprotein hormone receptor family (the LH and thyroid-stimulating hormone receptors), it is not known whether the follicle-stimulating hormone receptor (FSHR) is palmitoylated and what are the effects of abolishing its potential palmitoylation sites.