Differences in signal activation by LH and hCG are mediated by the LH/CG receptor’s extracellular hinge region

The human lutropin (hLH)/choriogonadotropin (hCG) receptor (LHCGR) can be activated by binding two slightly different gonadotropic glycoprotein hormones, choriogonadotropin (CG) – secreted by the placenta, and lutropin (LH) – produced by the pituitary. They induce different signaling profiles at the LHCGR. This cannot be explained by binding to the receptor’s leucine-rich- repeat domain (LRRD), as this binding is similar for the two hormones. We therefore speculate that there are previously unknown differences in the hormone/receptor interaction at the extracellular hinge region, which might help to understand functional differences between the two hormones. We have therefore performed a detailed study of the binding and action of LH and CG at the LHCGR hinge region. We focused on a primate-specific additional exon in the hinge region, which is located between LRRD and the serpentine domain. The segment of the hinge region encoded by exon10 was previously reported to be only relevant to hLH signaling, as the exon10-deletion receptor exhibits decreased hLH signaling, but unchanged hCG signaling. We designed an advanced homology model of the hormone/LHCGR complex, followed by experimental characterization of relevant fragments in the hinge region. In addition, we examined predictions of a helical exon10-encoded conformation by block-wise polyalanine (helix supporting) mutations. These helix preserving modifications showed no effect on hormone-induced signaling. However, introduction of a structure-disturbing double-proline mutant LHCGR-Q303P/E305P within the exon10-helix has, in contrast to exon10-deletion, no impact on hLH, but only on hCG signaling. This opposite effect on signaling by hLH and hCG can be explained by distinct sites of hormone interaction in the hinge region. In conclusion, our analysis provides details of the differences between hLH- and hCG-induced signaling that are mainly determined in the L2-beta loop of the hormones and in the hinge region of the receptor

Endothelin potentiates TRPV1 via ETA receptor-mediated activation of protein kinase C

© 2007 Plant et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

Mass-spectrometric identification of a novel angiotensin peptide in human plasma

Objective— Angiotensin peptides play a central role in cardiovascular physiology and pathology. Among these peptides, angiotensin II (Ang II) has been investigated most intensively. However, further angiotensin peptides such as Ang 1-7, Ang III, and Ang IV also contribute to vascular regulation, and may elicit additional, different, or even opposite effects to Ang II. Here, we describe a novel Ang II-related, strong vasoconstrictive substance in plasma from healthy humans and end-stage renal failure patients. Methods and Results— Chromatographic purification and structural analysis by matrix-assisted laser desorption/ionisation time-of-flight/time-of-flight (MALDI-TOF/TOF) revealed an angiotensin octapeptide with the sequence Ala-Arg-Val-Tyr-Ile-His-Pro-Phe, which differs from Ang II in Ala1 instead of Asp1. Des[Asp1]-[Ala1]-Ang II, in the following named Angiotensin A (Ang A), is most likely generated enzymatically. In the presence of mononuclear leukocytes, Ang II is converted to Ang A by decarboxylation of Asp1. Ang A has the same affinity to the AT1 receptor as Ang II, but a higher affinity to the AT2 receptor. In the isolated perfused rat kidney, Ang A revealed a smaller vasoconstrictive effect than Ang II, which was not modified in the presence of the AT2 receptor antagonist PD 123319, suggesting a lower intrinsic activity at the AT1 receptor. Ang II and Ang A concentrations in plasma of healthy subjects and end-stage renal failure patients were determined by matrix-assisted laser desorption/ionisation mass-analysis, because conventional enzyme immunoassay for Ang II quantification did not distinguish between Ang II and Ang A. In healthy subjects, Ang A concentrations were less than 20% of the Ang II concentrations, but the ratio Ang A / Ang II was higher in end-stage renal failure patients. Conclusion— Ang A is a novel human strong vasoconstrictive angiotensin-derived peptide, most likely generated by enzymatic transformation through mononuclear leukocyte-derived aspartate decarboxylase. Plasma Ang A concentration is increased in end-stage renal failure. Because of its stronger agonism at the AT2 receptor, Ang A may modulate the harmful effects of Ang II. In this study, a new angiotensin-peptide of human plasma is described, which is characterized as a strong AT2-receptor agonist

Improved Learning and Memory in Aged Mice Deficient in Amyloid β-Degrading Neutral Endopeptidase

BACKGROUND: Neutral endopeptidase, also known as neprilysin and abbreviated NEP, is considered to be one of the key enzymes in initial human amyloid-beta (Abeta) degradation. The aim of our study was to explore the impact of NEP deficiency on the initial development of dementia-like symptoms in mice. METHODOLOGY/PRINCIPAL FINDINGS: We found that while endogenous Abeta concentrations were elevated in the brains of NEP-knockout mice at all investigated age groups, immunohistochemical analysis using monoclonal antibodies did not detect any Abeta deposits even in old NEP knockout mice. Surprisingly, tests of learning and memory revealed that the ability to learn was not reduced in old NEP-deficient mice but instead had significantly improved, and sustained learning and memory in the aged mice was congruent with improved long-term potentiation (LTP) in brain slices of the hippocampus and lateral amygdala. Our data suggests a beneficial effect of pharmacological inhibition of cerebral NEP on learning and memory in mice due to the accumulation of peptides other than Abeta degradable by NEP. By conducting degradation studies and peptide measurements in the brain of both genotypes, we identified two neuropeptide candidates, glucagon-like peptide 1 and galanin, as first potential candidates to be involved in the improved learning in aged NEP-deficient mice. CONCLUSIONS/SIGNIFICANCE: Thus, the existence of peptides targeted by NEP that improve learning and memory in older individuals may represent a promising avenue for the treatment of neurodegenerative diseases

Novel Method for Quantifying AhR-Ligand Binding Affinities Using Microscale Thermophoresis.

The aryl hydrocarbon receptor (AhR) is a highly conserved cellular sensor of a variety of environmental pollutants and dietary-, cell- and microbiota-derived metabolites with important roles in fundamental biological processes. Deregulation of the AhR pathway is implicated in several diseases, including autoimmune diseases and cancer, rendering AhR a promising target for drug development and host-directed therapy. The pharmacological intervention of AhR processes requires detailed information about the ligand binding properties to allow specific targeting of a particular signaling process without affecting the remaining. Here, we present a novel microscale thermophoresis-based approach to monitoring the binding of purified recombinant human AhR to its natural ligands in a cell-free system. This approach facilitates a precise identification and characterization of unknown AhR ligands and represents a screening strategy for the discovery of potential selective AhR modulators

Late endosomal/lysosomal targeting and lack of recycling of the ligand-occupied endothelin B receptor

and Cy3-ET1 fluorescences were found in the perinuclear region, colocalized with fluorescent low density lipoproteins, a marker of the late endosomal/lysosomal pathway, but not with fluorescent transferrin, a marker of the recycling pathway. No dissociation of Cy3-ET1 from the receptor was seen within 4 h. Using 125 I-ET1 or Cy3-ET1, binding sites were again demonstrable at the cell surface within 2 h. The reappearance of binding sites was abolished by prior treatment of the cells with cycloheximide, an inhibitor of protein synthesis. The data demonstrate that the ligand-occupied ET B receptor is internalized; however, it does not recycle like most of the G protein-coupled receptors but is sorted to the late endosomal/lysosomal pathway in a manner similar to that of the family of proteaseactivated receptors

Identification and measurement of β-endorphin levels in the skin during induced hair growth in mice

We describe new and effective techniques for extracting proopiomelanocortin (POMC)-derived peptides from mammalian skin. Using this methodology (hot-acid extraction) and two independent HPLC-controlled RIA systems, we identify β-endorphin peptide in mammalian skin and demonstrate significant hair cycle-dependent fluctuations in both the skin concentration and the in situ expression pattern of β-endorphin (sebaceous glands) during the entire murine hair cycle. The observed anagen (growth phase)-associated increase in β-endorphin concentration and its decline during the follicle involution (catagen) or resting (telogen) phase raise the possibility of a regulatory function of this neuropeptide in cyclic changes of skin physiology