Cardiac CRFR1 Expression Is Elevated in Human Heart Failure and Modulated by Genetic Variation and Alternative Splicing.

Corticotropin-releasing factor (CRF) and the CRF-related peptides, urocortin (Ucn)-1, Ucn2, and Ucn3 signal through receptors CRFR1 and CRFR2 to restore homeostasis in response to stress. The Ucns exert potent cardioprotective effects and may have clinical utility in heart failure. To explore the activity of this system in the heart, we measured the levels of myocardial gene expression of the CRF/Ucn family of ligands/receptors and investigated genetic variation and alternative splicing of CRFR1 in 110 heart failure patients and 108 heart donors. Using quantitative real-time PCR, we detected CRFR1, CRFR2, CRF, Ucn1, Ucn2, and Ucn3 in all samples. CRFR2α was the most abundant receptor and Ucn3 the most abundant ligand, both in patients and donors. Compared with donors, cardiac expression of CRFR1, CRF, and Ucn3 was higher (P < .001) and CRFR2α lower (P = .012) in patients. In patients and donors, genetic variation within CRFR1, represented by the chromosome 17q21.31 inversion polymorphism, was associated with markedly higher CRFR1 expression (P < .001), making CRFR1 and CRFR2α expression almost equivalent in some patients. A novel, truncated splice variant of CRFR1, designated CRFR1j, was identified and shown to exert a dominant-negative effect on CRFR1 signaling in vitro. The novel variant was expressed in a greater proportion of patients (60%) than donors (3%, P < .001). In summary, cardiac expression of CRFR1, CRF, and Ucn3 genes is elevated in heart failure and may contribute to the activation of the CRF/Ucn system in these patients. A common variant within the CRFR1 gene and a novel CRFR1 splice variant may modulate CRFR1 expression and signaling.

Characterization of a Pachymedusa dacnicolor-Sauvagine analog as a new high-affinity radioligand for corticotropin-releasing factor receptor studies.

The corticotropin-releasing factor (CRF) peptide family comprises the mammalian peptides CRF and the urocortins as well as frog skin sauvagine and fish urophyseal urotensin. Advances in understanding the roles of the CRF ligand family and associated receptors have often relied on radioreceptor assays using labeled CRF ligands. These assays depend on stable, high-affinity CRF analogs that can be labeled, purified, and chemically characterized. Analogs of several of the native peptides have been used in this context, most prominently including sauvagine from the frog Phyllomedusa sauvageii (PS-Svg). Because each of these affords both advantages and disadvantages, new analogs with superior properties would be welcome. We find that a sauvagine-like peptide recently isolated from a different frog species, Pachymedusa dacnicolor (PD-Svg), is a high-affinity agonist whose radioiodinated analog, [(125)ITyr(0)-Glu(1), Nle(17)]-PD-Svg, exhibits improved biochemical properties over those of earlier iodinated agonists. Specifically, the PD-Svg radioligand binds both CRF receptors with comparably high affinity as its PS-Svg counterpart, but detects a greater number of sites on both type 1 and type 2 receptors. PD-Svg is also ∼10 times more potent at stimulating cAMP accumulation in cells expressing the native receptors. Autoradiographic localization using the PD-Svg radioligand shows robust specific binding to rodent brain and peripheral tissues that identifies consensus CRF receptor-expressing sites in a greater number and/or with greater sensitivity than its PS-Svg counterpart. We suggest that labeled analogs of PD-Svg may be useful tools for biochemical, structural, pharmacological, and anatomic studies of CRF receptors.

Expression and functional characterization of membrane-integrated mammalian corticotropin releasing factor receptors 1 and 2 in Escherichia coli.

Corticotropin-Releasing Factor Receptors (CRFRs) are class B1 G-protein-coupled receptors, which bind peptides of the corticotropin releasing factor family and are key mediators in the stress response. In order to dissect the receptors' binding specificity and enable structural studies, full-length human CRFR1α and mouse CRFR2ß as well as fragments lacking the N-terminal extracellular domain, were overproduced in E. coli. The characteristics of different CRFR2ß-PhoA gene fusion products expressed in bacteria were found to be in agreement with the predicted ones in the hepta-helical membrane topology model. Recombinant histidine-tagged CRFR1α and CRFR2ß expression levels and bacterial subcellular localization were evaluated by cell fractionation and Western blot analysis. Protein expression parameters were assessed, including the influence of E. coli bacterial hosts, culture media and the impact of either PelB or DsbA signal peptide. In general, the large majority of receptor proteins became inserted in the bacterial membrane. Across all experimental conditions significantly more CRFR2ß product was obtained in comparison to CRFR1α. Following a detergent screen analysis, bacterial membranes containing CRFR1α and CRFR2ß were best solubilized with the zwitterionic detergent FC-14. Binding of different peptide ligands to CRFR1α and CRFR2ß membrane fractions were similar, in part, to the complex pharmacology observed in eukaryotic cells. We suggest that our E. coli expression system producing functional CRFRs will be useful for large-scale expression of these receptors for structural studies.

Endogenous betaglycan is essential for high-potency inhibin antagonism in gonadotropes.

Inhibins are endocrine hormones that regulate gametogenesis and reproduction through a negative feedback loop with FSH. Inhibin action involves antagonism of signaling by activin or other TGFbeta family ligands. In transfection assays, antagonism by inhibin can be potentiated by betaglycan, a coreceptor for selected TGFbeta family ligands. We tested whether betaglycan is an obligate inhibin coreceptor through disruption of betaglycan function by RNA interference-mediated knockdown and immunoneutralization. Betaglycan knockdown and anti-betaglycan IgG each independently prevented inhibin-A binding to betaglycan and reversed functional effects of transfected betaglycan. Neither betaglycan immunoneutralization nor knockdown affected activin responsiveness in cell lines or in rat anterior pituitary cultures. Betaglycan knockdown decreased the potency of inhibin antagonism of activin-induced FSH secretion in primary gonadotropes. Similarly, anti-betaglycan IgG decreased the potency of inhibin antagonism in primary gonadotropes in a dose-dependent manner, with a reduction in the sensitivity to inhibin-A of greater than 1000-fold. These data establish that betaglycan is an endogenous inhibin coreceptor required for high-sensitivity inhibin antagonism of activin signaling in rat anterior pituitary gonadotropes.

Identification of distinct inhibin and transforming growth factor beta-binding sites on betaglycan: functional separation of betaglycan co-receptor actions.

Betaglycan is a co-receptor that mediates signaling by transforming growth factor beta (TGFbeta) superfamily members, including the distinct and often opposed actions of TGFbetas and inhibins. Loss of betaglycan expression, or abrogation of betaglycan function, is implicated in several human and animal diseases, although both betaglycan actions and the ligands involved in these disease states remain unclear. Here we identify a domain spanning amino acids 591-700 of the betaglycan extracellular domain as the only inhibin-binding region in betaglycan. This binding site is within the betaglycan ZP domain, but inhibin binding is not integral to the ZP motif of other proteins. We show that the inhibin and TGFbeta-binding residues of this domain overlap and identify individual amino acids essential for binding of each ligand. Mutation of Val614 to Tyr abolishes both inhibin and TGFbeta binding to this domain. Full-length betaglycan V614Y, and other mutations, retain TGFbeta binding activity via a distinct site, but are unable to bind inhibin-A. These betaglycan mutants fail to mediate inhibin antagonism of activin signaling but can present TGFbeta to TbetaRII. Separating the co-receptor actions of betaglycan toward inhibin and TGFbeta will allow the clarification of the role of betaglycan in disease states such as renal cell carcinoma and endometrial adenocarcinoma.

A soluble mouse brain splice variant of type 2alpha corticotropin-releasing factor (CRF) receptor binds ligands and modulates their activity.

Peptides of the corticotropin-releasing factor (CRF) family signal through the activation of two receptors, CRF receptor type 1 (CRFR1) and type 2 (CRFR2), both of which exist as multiple splice variants. We have identified a cDNA from mouse brain encoding a splice variant, soluble CRFR2alpha (sCRFR2alpha), in which exon 6 is deleted from the gene encoding CRFR2alpha. Translation of this isoform produces a predicted 143-aa soluble protein. The translated protein includes a majority of the first extracellular domain of the CRFR2alpha followed by a unique 38-aa hydrophilic C terminus resulting from a frame shift produced by deletion of exon 6. By using RT-PCR and Southern hybridization, the relative mRNA expression levels of full-length (seven transmembrane domains) CRFR2alpha and the soluble form (sCRFR2alpha) in the mouse brain were measured with a single reaction. The results demonstrate high levels of expression of sCRFR2alpha in the olfactory bulb, cortex, and midbrain regions. A rabbit antiserum raised against a synthetic peptide fragment encoding the unique C terminus revealed specific sCRFR2alpha immunoreactivity in mouse brain slices by immunohistochemistry and in extracts of brain regions by RIA. Interestingly, the sCRFR2alpha immunoreactivity distribution closely approximated that of CRFR1 expression in rodent brain. A protein corresponding to sCRFR2alpha, expressed and purified from either mammalian or bacterial cell systems, binds several CRF family ligands with low nanomolar affinities. Furthermore, the purified sCRFR2alpha protein inhibits cellular responses to CRF and urocortin 1. These data support a potential role of the sCRFR2alpha protein as a possible biological modulator of CRF family ligands.