Non-HLA antibodies targeting angiotensin II Type 1 receptor and endothelin-1 Type A receptors induce endothelial injury via ß2-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, ß-arrestins, and the major regulator of cell growth, PI3K/mTOR signaling, in impaired endothelial repair. To test this, human microvascular endothelial cells were treated with AT1R/ETAR antibodies isolated from patients with kidney transplant vasculopathy. These antibodies activated both mTOR complexes via AT1R and ETAR in a PI3K-dependent and ERK-independent manner. The mTOR inhibitor, rapamycin, completely abolished activation of mTORC1 and mTORC2 after long-term treatment with receptor antibodies. Imaging studies revealed that ß2- but not ß1-arrestin was recruited to ETAR in response to ET-1 and patient antibodies but not with antibodies isolated from healthy individuals. Silencing of ß2-arrestin by siRNA transfection significantly reduced ERK1/2 and mTORC2 activation. Non-HLA antibodies impaired endothelial repair by AT1R- and ETAR-induced mTORC2 signaling. Thus, we provide evidence that functional AT1R/ETAR antibodies induce ERK1/2 and mTOR signaling involving ß2-arrestin in human microvascular endothelium. Hence, our data may provide a translational rationale for mTOR inhibitors in combination with receptor blockers in patients with non-HLA receptor recognizing antibodies.

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. Screening for small molecule inhibitors impairing its function has thus been notoriously difficult. However, such translocation inhibitors may not only be valuable tools for cell biology, but may also represent novel anticancer drugs, given that cancer cells heavily depend on efficient protein translocation into the ER to support their fast growth. In this review, different inhibitors of protein translocation will be discussed, and their specific mode of action will be compared. In addition, recently published screening strategies for small molecule inhibitors targeting the whole SRP-Sec61 targeting/translocation pathway will be summarized. Of note, slightly modified assays may be used in the future to screen for substances affecting SecYEG, the bacterial ortholog of the Sec61 complex, in order to identify novel antibiotic drugs.

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. To screen for these proteins, we employed stable isotope labeling by amino acids in cell culture technique in combination with quantitative MS on CADA-treated human T-lymphoid SUP-T1 cells expressing high levels of huCD4. In line with our previous reports, our current proteomic analysis (data available via ProteomeXchange with identifier PXD027712) demonstrated that only a very small subset of proteins is depleted by CADA. Our data also confirmed that cellular expression of both huCD4 and sortilin are affected by CADA treatment of SUP-T1 cells. Furthermore, three additional targets for CADA are identified, namely, endoplasmic reticulum lectin 1 (ERLEC1), inactive tyrosine-protein kinase 7 (PTK7), and DnaJ homolog subfamily C member 3 (DNAJC3). Western blot and flow cytometry analysis of ERLEC1, PTK7, and DNAJC3 protein expression validated susceptibility of these substrates to CADA, although with varying degrees of sensitivity. Additional cell-free in vitro translation/translocation data demonstrated that the new substrates for CADA carry cleavable SPs that are targets for the cotranslational translocation inhibition exerted by CADA. Thus, our quantitative proteomic analysis demonstrates that ERLEC1, PTK7, and DNAJC3 are validated additional substrates of CADA; however, huCD4 remains the most sensitive integral membrane protein for the endoplasmic reticulum translocation inhibitor CADA. Furthermore, to our knowledge, CADA is the first compound that specifically interferes with only a very small subset of SPs and does not affect signal anchor sequences.

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. The activated TSHR but also the cross-interacting insulin-like growth factor 1 receptor (IGF-1R) contribute to this issue. The TSHR is a heptahelical G-protein-coupled receptor, whereas the IGF-1R is a receptor tyrosine kinase. Despite these fundamental structural differences, both receptors are phosphorylated by G-protein receptor kinases, which enables ß-arrestin binding. Arrestins mediate receptor internalization and also activate the mitogen-activated protein kinase pathway. Moreover, emerging results suggest that arrestin plays a critical role in the cross-interaction of the TSHR and the IGF-1R either in their common signaling pathway and/or during an indirect or potential TSHR/IGF-1R interaction. In this review, novel pharmacological strategies with allosteric small-molecule modulators to treat GO and GD on the level of the TSHR and/or the TSHR/IGF-1R cross-interaction will be discussed. Moreover, monoclonal antibody approaches targeting the TSHR or the IGF-1R and thereby preventing activation of either receptor will be presented. Another chapter addresses the immunomodulation to treat GO using TSHR-derived peptides targeting the human leukocyte antigen DR isotope (HLA-DR), which is a feasible approach to tackle GO, since HLA-DR and TSHR are overexpressed in orbital tissues of GO patients.

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). From the inhibitory effects of NAM S37a on: 1) chimeras with swapped ectodomain, 2) stepwise N-terminal truncations, 3) distinct constitutively active mutations distributed across the hinge region and ECL, but not across the TMD, we conclude that S37a binds at the ectodomain/TMD interface, between the converging helix, ECL1, and the IA. This is also supported by the noncompetitive inhibition of PAM-C2-activation by S37a in the TSHR-TMD construct lacking the ectodomain. Mutagenesis studies on the IA and ECL were guided by our refined model of the ectodomain/TMD interface and indicate an interaction with the TSHR-specific residues E404 (preceding IA) and H478 (ECL1). At this new allosteric interaction site, NAM S37a blocks both TSH- and PAM-induced activation of the TSHR. Our refined models, mutations, and new allosteric binding pocket helped us to gain more detailed insights into the intramolecular course of TSHR activation at the ectodomain/TMD interface, including the delocalization of the converging helix and rearrangement of the conformation of IA. These changes are embedded between the ECL and cooperatively trigger active conformations of TMD. SIGNIFICANCE STATEMENT: The intramolecular activation mechanisms of the TSHR appear to be distinct from those of other G protein-coupled receptors, as the TSHR has a uniquely large N-terminal ectodomain that includes the hormone binding site and an internal agonist sequence. We present new molecular and structural insights into the interface between ectodomain and transmembrane domain in the TSHR, as well as the transfer of activation to the transmembrane domain. This knowledge is critical for understanding activation or inhibition of the receptor by allosteric ligands. We have identified a new allosteric antagonist binding pocket that is located exactly at this interface and possesses specific features that may allow the generation of potent highly TSHR-selective drugs, of potential value for the treatment of Graves' orbitopathy.

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. Stereo-selective synthesis of the screening hits led to the molecule S37, which contains seven chiral centers. Enantiomeric separation of the molecule S37 resulted in the enantiopure molecule S37a-a micro-molar antagonist of thyrotropin-induced cyclic adenosine monophosphate accumulation in HEK 293 cells expressing the TSHR. RESULTS: The unique rigid bent shape of molecule S37a may mediate the observed high TSHR selectivity. Most importantly, the closely related follitropin and lutropin receptors were not affected by this compound. S37a not only inhibits the TSHR activation by thyrotropin itself but also activation by monoclonal TSAb M22 (human), KSAb1 (murine), and the allosteric small-molecule agonist C2. Disease-related ex vivo studies in HEK 293 cells expressing the TSHR showed that S37a also inhibits cyclic adenosine monophosphate formation by oligoclonal TSAb, which are highly enriched in GO patients' sera. Initial in vivo pharmacokinetic studies revealed no toxicity of S37a and a remarkable 53% oral bioavailability in mice. CONCLUSION: In summary, a novel highly selective inhibitor for the TSHR is presented, which has promising potential for further development for the treatment of GO.

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. The Sec61 complex has no enzymatic activity, is expressed only intracellularly and is difficult to purify and to reconstitute. Screening for small molecule inhibitors impairing its functions is thus notoriously difficult. Such inhibitors may not only be valuable tools for cell biology, they may also represent novel anti-tumor drugs. Here we have developed a two-step, sequential screening assay for inhibitors of the whole SRP-Sec61 targeting/translocation pathway which might include molecules affecting Sec61 complex functions. The resulting hit compounds were analyzed using a whole cell biosynthesis assay and a cell free transcription/translation/translocation assay. Using this methodology, we identified novel compounds inhibiting this pathway. Following structure-based back screening, one of these substances was analyzed in more detail and we could show that it indeed impairs translocation at the level of the Sec61 complex. A slightly modified methodology may be used in the future to screen for substances affecting SecYEG, the bacterial ortholog of the Sec61 complex in order to derive novel antibiotic drugs.

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. Indeed, we demonstrate strong interaction between both the proteins which causes a suppressed activation of Gq/11 by TSH-stimulated TSHR. Thus, we provide not only evidence for a novel interaction between the TSHR and MCT8, but could also prove this interaction on a single molecule level. Moreover, this interaction forces biased signaling at the TSHR. These results are of general interest for both the GPCR and the MFS research fields.

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. Such cross-inhibition may indicate heterodimerization of these receptors. Therefore, this study investigated the molecular and functional interplay between MAS and the AT2R. Molecular interactions were assessed by fluorescence resonance energy transfer and by cross correlation spectroscopy in human embryonic kidney-293 cells transfected with vectors encoding fluorophore-tagged MAS or AT2R. Functional interaction of AT2R and MAS was studied in astrocytes with CX3C chemokine receptor-1 messenger RNA expression as readout. Coexpression of fluorophore-tagged AT2R and MAS resulted in a fluorescence resonance energy transfer efficiency of 10.8 ± 0.8%, indicating that AT2R and MAS are capable to form heterodimers. Heterodimerization was verified by competition experiments using untagged AT2R and MAS. Specificity of dimerization of AT2R and MAS was supported by lack of dimerization with the transient receptor potential cation channel, subfamily C-member 6. Dimerization of the AT2R was abolished when it was mutated at cysteine residue 35. AT2R and MAS stimulation with the respective agonists, Compound 21 or angiotensin-(1-7), significantly induced CX3C chemokine receptor-1 messenger RNA expression. Effects of each agonist were blocked by an AT2R antagonist (PD123319) and also by a MAS antagonist (A-779). Knockout of a single of these receptors made astrocytes unresponsive for both agonists. Our results suggest that MAS and the AT2R form heterodimers and that-at least in astrocytes-both receptors functionally depend on each other.

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. We report a capture compound (CC)-based strategy to target and identify GPCRs directly from living cells. We synthesized CCs with sertindole attached to the CC scaffold in different orientations to target the dopamine D2 receptor (DRD2) heterologously expressed in HEK 293 cells. The structure-activity relationship of sertindole for DRD2 binding was reflected in the activities of the sertindole CCs in radioligand displacement, cell-based assays, and capture compound mass spectrometry (CCMS). The activity pattern was rationalized by molecular modelling. The most-active CC showed activities very similar to that of unmodified sertindole. A concentration of DRD2 in living cells well below 100 fmol used as an experimental input was sufficient for unambiguous identification of captured DRD2 by mass spectrometry. Our new CCMS workflow broadens the arsenal of chemoproteomic technologies to close a critical gap for the comprehensive characterization of drug-protein interactions.

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. Most GPCRs possess signal sequences which form part of the mature proteins, the so called signal anchor sequences (usually transmembrane domain 1). The CRF receptors possess instead signal sequences at their extreme N tails which were thought to be cleaved off following integration of the receptors into the ER membrane (signal peptides, SPs, also called cleaved signal sequences). Recent work, however, showed that not all subtypes of CRF receptors stick to this rule. Whereas the corticotropin-releasing factor receptor type 1 (CRF1R) and the corticotropin-releasing factor receptor type 2b (CRF2(b)R) possess conventional SPs which are indeed cleaved off following ER insertion, the SP of the cortictropin-releasing factor receptor type 2a (CRF2(a)R) remains uncleaved. It forms a unique N-terminal domain (pseudo signal peptide, PSP) which has surprising functions beyond the ER level. Its presence not only influences expression levels at the plasma membrane but also receptor homodimerisation and, as a consequence, G protein selectivity. In this mini-review, we summarize the progress in understanding the functions of SPs of CRF receptors. Recent data also allow deriving hypotheses for a physiological significance of these sequences.

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'-T2 and T3, but not rT3 and T4. We utilized LAT2 mutations (Y130A, N133S, F242W) that increase 3,3'-T2 import and focus here on import and export capacity for AA, T4, T3, BCH and derivatives thereof to delineate molecular features. Transport studies and analysis of competitive inhibition of import by radiolabelled TH and AA were performed in Xenopus laevis oocytes. Only Y130A, a pocket widening mutation, enabled import for T4 and increased it for T3. Mutant F242W showed increased 3,3'-T2 import but no import rates for other TH derivatives. No export was detected for any TH by LAT2-wild type (WT). Mutations Y130A and N133S enabled only the export of 3,3'-T2, while N133S also increased AA export. Thus, distinct molecular LAT2-features determine bidirectional AA transport but only an unidirectional 3,3'-T2 and T3 import.

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. LAT2) and a heavy chain (CD98), which is essential for their cell surface expression and functionality. The specificity of Lat2 for thyroid hormones and their metabolites and its role in their transport was not fully clear. This fact motivated us to establish a cell system to elucidate the uptake of thyroid hormones and their metabolites by mouse Lat2. The coinjection of cRNA coding for Lat2 and CD98 into Xenopus laevis oocytes resulted in a markedly increased level of 3,3'-diiodo-L-thyronine (3,3'-T2) and to some extent also enhanced T3 transport. To gain insight into properties of thyroid hormones and their metabolites transported by Lat2, we inhibited 3,3'-T2 uptake by various iodothyronine derivatives. T1 and T2 derivatives as well as 2-aminobicyclo-[2, 2,1]-heptane-2-carboxylic acid strongly competed with 3,3'-T2 uptake. In addition, we performed T2 uptake measurements with the thyroid hormone-specific transporter MCT8. For both Lat2 and MCT8, Km values in a low micromolar range were calculated. We demonstrated that oocytes are a suitable system for thyroid hormone transport studies mediated by Lat2. Our data indicates that Lat2 compared to other thyroid hormone transporters prefers 3,3'-T2 as the substrate. Thus, Lat2 might contribute to the availability of thyroid hormone by importing and/or exporting 3,3'-T2, which is generated either by T3 inactivation or by rapid deiodinase 1-mediated rT3 degradation.

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. Testis biopsy showed profound LCH and absent germinal line elements (Sertoli-only syndrome). The sequence analysis of the LHCGR gene showed the presence of a compound heterozygosity, being one variation, c.1847C>A p.S616Y, already described in association to Hypergonadotropic Hypogonadism, and the other, c.29 C>T p.L10P, a new identified variant in the putative signal peptide (SP) of LHCGR. Functional and structural studies provide first evidence that LHCGR have a functional and cleavable SP required for receptor biogenesis. Moreover, we demonstrate the pathogenic role of the novel p.L10P allelic variant, which has to be considered a loss-of-function mutation significantly contributing, in compound heterozygosity with p.S616Y, to the LCH type 2 observed in our patient.

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. About 5-10% of the GPCRs contain N-terminal signal peptides; the vast majority possesses signal anchor sequences. The reason why only a subset of GPCRs require signal peptides for ER targeting/insertion was addressed in the past decade by a limited number of studies indicating that the presence of signal peptides facilitates N-tail translocation at the ER membrane. Interestingly, recent work showed that signal peptides of GPCRs do not only serve "classical" functions in the early secretory pathway. Uncleaved pseudo signal peptides may regulate receptor densities in the plasma membrane, receptor dimerization, and G protein coupling selectivity. On the other hand, even cleaved and released peptides may have post-ER functions. In this review, we summarize the current knowledge about cleavable signal peptides of GPCRs and address also the question whether these sequences may serve as future drug targets in pharmacology.

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. Our Lat2 homology model is based on 2 crystal structures of the homologous 12-transmembrane helix transporters arginine/agmatine antiporter and amino acid/polyamine/organocation transporter. Model-driven mutagenesis of residues lining an extracellular recognition site and a TH-traversing channel identified 9 sensitive residues. Using Xenopus laevis oocytes as expression system, we found that side chain shortening (N51S, N133S, N248S, and Y130A) expanded the channel and increased 3,3'-T2 transport. Side chain enlargements (T140F, Y130R, and I137M) decreased 3,3'-T2 uptake, indicating channel obstructions. The opposite results with mutations maintaining (F242W) or impairing (F242V) uptake suggest that F242 may have a gating function. Competitive inhibition studies of 14 TH-like compounds revealed that recognition by Lat2 requires amino and carboxylic acid groups. The size of the adjacent hydrophobic group is restricted. Bulky substituents in positions 3 and 5 of the tyrosine ring are allowed. The phenolic ring may be enlarged, provided that the whole molecule is flexible enough to fit into the distinctly shaped TH-traversing channel of Lat2. Taken together, the next Lat2 features were identified 1) TH recognition site; 2) TH-traversing channel in the center of Lat2; and 3) switch site that potentially facilitates intracellular substrate release. Together with identified substrate features, these data help to elucidate the molecular mechanisms and role of Lat2 in T2 transport.

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. how many proteins are sensitive. Moreover, a consensus motif in signal sequences mediating cotransin sensitivity has yet not been described. To address these questions, we performed a proteomic study using cotransin-treated human hepatocellular carcinoma cells and the stable isotope labelling by amino acids in cell culture technique in combination with quantitative mass spectrometry. We used a saturating concentration of cotransin (30 micromolar) to identify also less-sensitive proteins and to discriminate the latter from completely resistant proteins. We found that the biosynthesis of almost all secreted proteins was cotransin-sensitive under these conditions. In contrast, biosynthesis of the majority of the integral membrane proteins was cotransin-resistant. Cotransin sensitivity of signal sequences was neither related to their length nor to their hydrophobicity. Instead, in the case of signal anchor sequences, we identified for the first time a conformational consensus motif mediating cotransin sensitivity.

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. To date, equilibrium measurements were restricted to the plasma membrane due to experimental limitations. We have addressed the question as to where this equilibrium is established for the corticotropin-releasing factor receptor type 1. By developing a novel approach to analyze single molecule fluorescence cross-correlation spectroscopy data for intracellular membrane compartments, we show that the corticotropin-releasing factor receptor type 1 has a specific monomer/dimer equilibrium that is already established in the endoplasmic reticulum (ER). It remains constant at the plasma membrane even following receptor activation. Moreover, we demonstrate for seven additional GPCRs that they are expressed in specific but substantially different monomer/dimer ratios. Although it is well known that proteins may dimerize in the ER in principle, our data show that the ER is also able to establish the specific monomer/dimer ratios of GPCRs, which sheds new light on the functions of this compartment.

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. In the past, the green fluorescent protein and its derivatives have been widely used as fluorescent tags. More recently, it was reported that photoconvertible fluorescent proteins (PCFPs) such as Kaede or Kikume green-red protein could also be used as fluorescent tags for GPCRs. These proteins have the obvious advantage that their fluorescence can be switched once the GPCR of interest has reached a specific subcellular compartment. Here, we summarize the recent progress for live cell imaging of GPCRs using these PCFPs for trafficking, biosynthesis, and protein/protein interaction studies.

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. Exon 10 encodes 27 amino acids within the hinge region, which is an extracellular segment that is important for signaling and hormone interaction. Initially, we assumed that the lack of exon 10 might disturb intermolecular trans-activation of hLH, a mechanism that has been reported for hCG at hLHR. Coexpression of signaling-deficient hLHR and binding-deficient hLHR can be used to examine the mechanisms of receptor signaling, in particular intermolecular cooperation and intramolecular cis-activation. Therefore, hLHR-delExon10 was combined with the hLHR Lys605→Glu mutant, in which signaling is abolished, and the hLHR mutant Cys131→Arg, in which binding is deficient. We found that hCG signaling was partially rescued, indicating trans-activation. However, the hLH signal could not be restored via forced trans-activation with any construct. Fluorescence cross-correlation spectroscopy detected oligomerization in all combinations, indicating that these functional differences cannot be explained by monomerization of hLHR-delExon10. Thus, our data demonstrate not only that the different behavior of hLH at hLHR-delExon10 is unlikely to be related to modified intermolecular receptor activation, but also that hLH may exclusively stimulate the targeted hLHR by cis-activation, whereas hCG is also capable of inducing trans-activation.