Inhibitors of the Sec61 Complex and Novel High Throughput Screening Strategies to Target the Protein Translocation Pathway.

Proteins targeted to the secretory pathway start their intracellular journey by being transported across biological membranes such as the endoplasmic reticulum (ER). A central component in this protein translocation process across the ER is the Sec61 translocon complex, which is only intracellularly expressed and does not have any enzymatic activity. In addition, Sec61 translocon complexes are difficult to purify and to reconstitute.

Non-HLA antibodies targeting angiotensin II Type 1 receptor and endothelin-1 Type A receptors induce endothelial injury via β2-arrestin link to mTOR pathway.

Functional non-HLA antibodies (antibodies to non-human leukocyte antigens) targeting the G protein-coupled receptors angiotensin II type 1 receptor (AT1R) and endothelin-1 type A receptor (ETAR) are implicated in the pathogenesis of transplant vasculopathy. While ERK signaling (a regulator of cell growth) may represent a general cellular response to agonist stimulation, the molecular link between receptor stimulation and development of vascular obliteration has not been fully established. Here we hypothesize involvement of the versatile adaptor proteins, β-arrestins, and the major regulator of cell growth, PI3K/mTOR signaling, in impaired endothelial repair.

A Proteomic Study on the Membrane Protein Fraction of T Cells Confirms High Substrate Selectivity for the ER Translocation Inhibitor Cyclotriazadisulfonamide.

Cyclotriazadisulfonamide (CADA) inhibits the cotranslational translocation of type I integral membrane protein human CD4 (huCD4) across the endoplasmic reticulum in a signal peptide (SP)-dependent way. Previously, sortilin was identified as a secondary substrate for CADA but showed reduced CADA sensitivity as compared with huCD4. Here, we performed a quantitative proteomic study on the crude membrane fraction of human T-cells to analyze how many proteins are sensitive to CADA.

Modulating TSH Receptor Signaling for Therapeutic Benefit.

Autoimmune thyroid-stimulating antibodies are activating the thyrotropin receptor (TSHR) in both the thyroid and the eye, but different molecular mechanisms are induced in both organs, leading to Graves' disease (GD) and Graves' orbitopathy (GO), respectively. Therapy with anti-thyroid drugs to reduce hyperthyroidism (GD) by suppressing the biosynthesis of thyroid hormones has only an indirect effect on GO, since it does not causally address pathogenic TSHR activation itself. GO is thus very difficult to treat.

Thyrotropin Receptor: Allosteric Modulators Illuminate Intramolecular Signaling Mechanisms at the Interface of Ecto- and Transmembrane Domain.

The large TSH-bound ectodomain of the thyrotropin receptor (TSHR) activates the transmembrane domain (TMD) indirectly via an internal agonist (IA). The ectodomain/TMD interface consists of a converging helix, a Cys-Cys-bridge-linked IA, and extracellular loops (ECL). To investigate the intramolecular course of molecular activation, especially details of the indirect activation, we narrowed down allosteric inhibition sites of negative allosteric modulator (NAM) by mutagenesis, homology modeling, and competition studies with positive allosteric modulator (PAM).

Use of a sequential high throughput screening assay to identify novel inhibitors of the eukaryotic SRP-Sec61 targeting/translocation pathway.

The SRP-Sec61 targeting/translocation pathway of eukaryotic cells targets nascent protein chains to the membrane of the endoplasmic reticulum. Using this machinery, secretory proteins are translocated across this membrane whereas membrane proteins are integrated into the lipid bilayer. One of the key players of the pathway is the protein-conducting Sec61 (translocon) complex of the endoplasmic reticulum.

A New Highly Thyrotropin Receptor-Selective Small-Molecule Antagonist with Potential for the Treatment of Graves' Orbitopathy.

Background: The thyrotropin receptor (TSHR) is the target for autoimmune thyroid stimulating antibodies (TSAb) triggering hyperthyroidism. Whereas elevated thyroid hormone synthesis by the thyroid in Graves' disease can be treated by antithyroid agents, for the pathogenic activation of TSHR in retro-orbital fibroblasts of the eye, leading to Graves' orbitopathy (GO), no causal TSHR directed therapy is available.

Methods: Due to the therapeutic gap for severe GO, TSHR inhibitors were identified by high-throughput screening in Chinese hamster ovary cells expressing the TSHR.

Evidence of G-protein-coupled receptor and substrate transporter heteromerization at a single molecule level.

G-protein-coupled receptors (GPCRs) can constitute complexes with non-GPCR integral membrane proteins, while such interaction has not been demonstrated at a single molecule level so far. We here investigated the potential interaction between the thyrotropin receptor (TSHR) and the monocarboxylate transporter 8 (MCT8), a member of the major facilitator superfamily (MFS), using fluorescence cross-correlation spectroscopy (FCCS). Both the proteins are expressed endogenously on the basolateral plasma membrane of the thyrocytes and are involved in stimulation of thyroid hormone production and release.

Targeting G Protein-Coupled Receptors by Capture Compound Mass Spectrometry: A Case Study with Sertindole.

Unbiased chemoproteomic profiling of small-molecule interactions with endogenous proteins is important for drug discovery. For meaningful results, all protein classes have to be tractable, including G protein-coupled receptors (GPCRs). These receptors are hardly tractable by affinity pulldown from lysates.

Evidence for Heterodimerization and Functional Interaction of the Angiotensin Type 2 Receptor and the Receptor MAS.

The angiotensin type 2 receptor (AT2R) and the receptor MAS are receptors of the protective arm of the renin-angiotensin system. They mediate strikingly similar actions. Moreover, in various studies, AT2R antagonists blocked the effects of MAS agonists and vice versa.

Functional Significance of the Signal Peptides of Corticotropin-Releasing Factor Receptors.

The corticotropin releasing factor (CRF) receptors belong to the large family of G proteincoupled receptors (GPCRs) and must be transported to the plasma membrane to function properly. The first step of the intracellular transport of GPCRs is their insertion into the membrane of the endoplasmic reticulum (ER). This process is mediated by the translocon complex of the ER membrane and the signal sequences of the receptors.

Molecular features of the L-type amino acid transporter 2 determine different import and export profiles for thyroid hormones and amino acids.

The L-type amino acid transporter 2 (LAT2) imports amino acids (AA) and also certain thyroid hormones (TH), e.g. 3,3'-T and T, but not rT and T.

Involvement of the L-Type Amino Acid Transporter Lat2 in the Transport of 3,3'-Diiodothyronine across the Plasma Membrane.

Thyroid hormones are transported across cell membranes by transmembrane transporter proteins, for example by members of the monocarboxylate transporter (MCT) and the L-type amino acid transporter (LAT) families. LATs consist of a light chain (e.g.

A new variant in signal peptide of the human luteinizing hormone receptor (LHCGR) affects receptor biogenesis causing leydig cell hypoplasia.

The human luteinizing hormone/chorionic gonadotropin receptor (LHCGR) plays a fundamental role in male and female reproduction. In males, loss-of-function mutations in LHCGR have been associated with distinct degrees of impairment in pre- and postnatal testosterone secretion resulting in a variable phenotypic spectrum, classified as Leydig cell hypoplasia (LCH) type 1 (complete LH resistance and disorder of sex differentiation) and type 2 (partial LH resistance with impaired masculinization and fertility). Here, we report the case of an adolescent who came to the pediatric endocrinologist at the age of 12 years old for micropenis and cryptorchidism.

N-Terminal Signal Peptides of G Protein-Coupled Receptors: Significance for Receptor Biosynthesis, Trafficking, and Signal Transduction.

Signal sequences play a key role during the first steps of the intracellular transport of G protein-coupled receptors (GPCRs). They are involved in targeting of the nascent chains to the membrane of the endoplasmic reticulum (ER) and initiate integration of the newly synthesized receptors into this compartment. Two classes of signal sequences are known: N-terminal signal peptides, which are usually cleaved-off following ER insertion and internal signal sequences, the so-called signal anchor sequences, which form part of the mature proteins.

Structural insights into thyroid hormone transport mechanisms of the L-type amino acid transporter 2.

Thyroid hormones (THs) are transported across cell membranes by different transmembrane transporter proteins. In previous studies, we showed marked 3,3'-diiodothyronine (3,3'-T2) but moderate T3 uptake by the L-type amino acid transporter 2 (Lat2). We have now studied the structure-function relationships of this transporter and TH-like molecules.

Defining a conformational consensus motif in cotransin-sensitive signal sequences: a proteomic and site-directed mutagenesis study.

The cyclodepsipeptide cotransin was described to inhibit the biosynthesis of a small subset of proteins by a signal sequence-discriminatory mechanism at the Sec61 protein-conducting channel. However, it was not clear how selective cotransin is, i.e.

The specific monomer/dimer equilibrium of the corticotropin-releasing factor receptor type 1 is established in the endoplasmic reticulum.

G protein-coupled receptors (GPCRs) represent the most important drug targets. Although the smallest functional unit of a GPCR is a monomer, it became clear in the past decades that the vast majority of the receptors form dimers. Only very recently, however, data were presented that some receptors may in fact be expressed as a mixture of monomers and dimers and that the interaction of the receptor protomers is dynamic.

Use of Kaede and Kikume green-red fusions for live cell imaging of G protein-coupled receptors.

The fusion of fluorescent proteins to G protein-coupled receptors (GPCRs) is an important tool to study, e.g., trafficking and protein interactions of these important drug targets.

Differences between lutropin-mediated and choriogonadotropin-mediated receptor activation.

The human lutropin/choriogonadotropin receptor (hLHR) for the gonadotropic hormones human luteinizing hormone (hLH; lutropin) and human choriogonadotropin (hCG) is crucial for normal sexual development and fertility. We aimed to unravel differences between the two hLHR hormones in molecular activation mechanisms at hLHR. We utilized a specific hLHR variant that lacks exon 10 (hLHR-delExon10), which maintains full cAMP signaling by hCG, but decreases hLH-induced receptor signaling, resulting in a pathogenic phenotype.

Molecular sampling of the allosteric binding pocket of the TSH receptor provides discriminative pharmacophores for antagonist and agonists.

The TSHR (thyrotropin receptor) is activated endogenously by the large hormone thyrotropin and activated pathologically by auto-antibodies. Both activate and bind at the extracellular domain. Recently, SMLs (small-molecule ligands) have been identified, which bind in an allosteric binding pocket within the transmembrane domain.

The Pseudo signal peptide of the corticotropin-releasing factor receptor type 2A prevents receptor oligomerization.

N-terminal signal peptides mediate the interaction of native proteins with the translocon complex of the endoplasmic reticulum membrane and are cleaved off during early protein biogenesis. The corticotropin-releasing factor receptor type 2a (CRF(2(a))R) possesses an N-terminal pseudo signal peptide, which represents a so far unique domain within the large protein family of G protein-coupled receptors (GPCRs). In contrast to a conventional signal peptide, the pseudo signal peptide remains uncleaved and consequently forms a hydrophobic extension at the N terminus of the receptor.

Live cell imaging of G protein-coupled receptors.

Live cell imaging experiments with G protein-coupled receptors (GPCRs) tagged with fluorescent fusion proteins were originally performed to study trafficking and subcellular location of these important drug targets. In the past decade, however, substantial progress came from improved imaging methods and from the cloning of novel fluorescent fusion proteins. Today, these methods allow to visualize not only GPCR interactions but also, e.

The protease-activated receptor 1 possesses a functional and cleavable signal peptide which is necessary for receptor expression.

The protease-activated receptor 1 (PAR1) is activated by thrombin cleavage releasing the physiologically-relevant parstatin peptide (residues 1-41). However, the actual length of parstatin was unclear since the receptor may also possess a cleavable signal peptide (residues 1-21) according to prediction programs. Here, we show that this putative signal peptide is indeed functional and removed from the PAR1 resolving the question of parstatin length.

Use of Kikume green-red fusions to study the influence of pharmacological chaperones on trafficking of G protein-coupled receptors.

In this study we demonstrate that the photoconvertible monomeric Kikume green-red (mKikGR) protein is suitable to study trafficking of G protein-coupled receptors. Taking mKikGR-tagged mutants of the vasopressin V(2) receptor (V(2)R) as models, we analyzed whether the V(2)R-specific pharmacological chaperone SR121463B influences receptor folding on a co- or post-translational level. Misfolded mKikGR-tagged V(2)Rs were completely photoconverted in the early secretory pathway yielding a red receptor population (already synthesized receptors) and an arising green receptor population (newly synthesized receptors).