@article{BalakrishnanHemmenChoudhuryetal.2022, author = {Balakrishnan, Ashwin and Hemmen, Katherina and Choudhury, Susobhan and Krohn, Jan-Hagen and Jansen, Kerstin and Friedrich, Mike and Beliu, Gerti and Sauer, Markus and Lohse, Martin J. and Heinze, Katrin G.}, title = {Unraveling the hidden temporal range of fast β2-adrenergic receptor mobility by time-resolved fluorescence}, series = {Communications Biology}, volume = {5}, journal = {Communications Biology}, number = {1}, doi = {10.1038/s42003-022-03106-4}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-301140}, year = {2022}, abstract = {G-protein-coupled receptors (GPCRs) are hypothesized to possess molecular mobility over a wide temporal range. Until now the temporal range has not been fully accessible due to the crucially limited temporal range of available methods. This in turn, may lead relevant dynamic constants to remain masked. Here, we expand this dynamic range by combining fluorescent techniques using a spot confocal setup. We decipher mobility constants of β\(_{2}\)-adrenergic receptor over a wide time range (nanosecond to second). Particularly, a translational mobility (10 µm\(^{2}\)/s), one order of magnitude faster than membrane associated lateral mobility that explains membrane protein turnover and suggests a wider picture of the GPCR availability on the plasma membrane. And a so far elusive rotational mobility (1-200 µs) which depicts a previously overlooked dynamic component that, despite all complexity, behaves largely as predicted by the Saffman-Delbr{\"u}ck model.}, language = {en} } @article{WoelfelSaetteleZechmeisteretal.2020, author = {W{\"o}lfel, Angela and S{\"a}ttele, Mathias and Zechmeister, Christina and Nikolaev, Viacheslov O. and Lohse, Martin J. and Boege, Fritz and Jahns, Roland and Boivin-Jahns, Val{\´e}rie}, title = {Unmasking features of the auto-epitope essential for β\(_1\)-adrenoceptor activation by autoantibodies in chronic heart failure}, series = {ESC Heart Failure}, volume = {7}, journal = {ESC Heart Failure}, number = {4}, doi = {10.1002/ehf2.12747}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-235974}, pages = {1830-1841}, year = {2020}, abstract = {Aims Chronic heart failure (CHF) can be caused by autoantibodies stimulating the heart via binding to first and/or second extracellular loops of cardiac β1-adrenoceptors. Allosteric receptor activation depends on conformational features of the autoantibody binding site. Elucidating these features will pave the way for the development of specific diagnostics and therapeutics. Our aim was (i) to fine-map the conformational epitope within the second extracellular loop of the human β\(_1\)-adrenoceptor (β1ECII) that is targeted by stimulating β\(_1\)-receptor (auto)antibodies and (ii) to generate competitive cyclopeptide inhibitors of allosteric receptor activation, which faithfully conserve the conformational auto-epitope. Methods and results Non-conserved amino acids within the β\(_1\)EC\(_{II}\) loop (compared with the amino acids constituting the ECII loop of the β\(_2\)-adrenoceptor) were one by one replaced with alanine; potential intra-loop disulfide bridges were probed by cysteine-serine exchanges. Effects on antibody binding and allosteric receptor activation were assessed (i) by (auto)antibody neutralization using cyclopeptides mimicking β1ECII ± the above replacements, and (ii) by (auto)antibody stimulation of human β\(_1\)-adrenoceptors bearing corresponding point mutations. With the use of stimulating β\(_1\)-receptor (auto)antibodies raised in mice, rats, or rabbits and isolated from exemplary dilated cardiomyopathy patients, our series of experiments unmasked two features of the β\(_1\)EC\(_{II}\) loop essential for (auto)antibody binding and allosteric receptor activation: (i) the NDPK\(^{211-214}\) motif and (ii) the intra-loop disulfide bond C\(^{209}\)↔C\(^{215}\). Of note, aberrant intra-loop disulfide bond C\(^{209}\)↔C\(^{216}\) almost fully disrupted the functional auto-epitope in cyclopeptides. Conclusions The conformational auto-epitope targeted by cardio-pathogenic β\(_1\)-receptor autoantibodies is faithfully conserved in cyclopeptide homologues of the β\(_1\)EC\(_{II}\) loop bearing the NDPK\(^{211-214}\) motif and the C\(^{209}\)↔C\(^{215}\) bridge while lacking cysteine C216. Such molecules provide promising tools for novel diagnostic and therapeutic approaches in β\(_1\)-autoantibodypositive CHF.}, language = {en} } @article{AgarwalYangRiceetal.2014, author = {Agarwal, Shailesh R. and Yang, Pei-Chi and Rice, Monica and Singer, Cherie A. and Nikolaev, Viacheslav O. and Lohse, Martin J. and Clancy, Colleen E. and Harvey, Robert D.}, title = {Role of Membrane Microdomains in Compartmentation of cAMP Signaling}, series = {PLOS ONE}, volume = {9}, journal = {PLOS ONE}, number = {4}, issn = {1932-6203}, doi = {10.1371/journal.pone.0095835}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-116673}, pages = {e95835}, year = {2014}, abstract = {Spatially restricting cAMP production to discrete subcellular locations permits selective regulation of specific functional responses. But exactly where and how cAMP signaling is confined is not fully understood. Different receptors and adenylyl cyclase isoforms responsible for cAMP production are not uniformly distributed between lipid raft and non-lipid raft domains of the plasma membrane. We sought to determine the role that these membrane domains play in organizing cAMP responses in HEK293 cells. The freely diffusible FRET-based biosensor Epac2-camps was used to measure global cAMP responses, while versions of the probe targeted to lipid raft (Epac2-MyrPalm) and non-raft (Epac2-CAAX) domains were used to monitor local cAMP production near the plasma membrane. Disruption of lipid rafts by cholesterol depletion selectively altered cAMP responses produced by raft-associated receptors. The results indicate that receptors associated with lipid raft as well as non-lipid raft domains can contribute to global cAMP responses. In addition, basal cAMP activity was found to be significantly higher in non-raft domains. This was supported by the fact that pharmacologic inhibition of adenylyl cyclase activity reduced basal cAMP activity detected by Epac2-CAAX but not Epac2-MyrPalm or Epac2-camps. Responses detected by Epac2-CAAX were also more sensitive to direct stimulation of adenylyl cyclase activity, but less sensitive to inhibition of phosphodiesterase activity. Quantitative modeling was used to demonstrate that differences in adenylyl cyclase and phosphodiesterase activities are necessary but not sufficient to explain compartmentation of cAMP associated with different microdomains of the plasma membrane.}, language = {en} } @article{KlenkHommersLohse2022, author = {Klenk, Christoph and Hommers, Leif and Lohse, Martin J.}, title = {Proteolytic cleavage of the extracellular domain affects signaling of parathyroid hormone 1 receptor}, series = {Frontiers in Endocrinology}, volume = {13}, journal = {Frontiers in Endocrinology}, issn = {1664-2392}, doi = {10.3389/fendo.2022.839351}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-262055}, year = {2022}, abstract = {Parathyroid hormone 1 receptor (PTH1R) is a member of the class B family of G protein-coupled receptors, which are characterized by a large extracellular domain required for ligand binding. We have previously shown that the extracellular domain of PTH1R is subject to metalloproteinase cleavage in vivo that is regulated by ligand-induced receptor trafficking and leads to impaired stability of PTH1R. In this work, we localize the cleavage site in the first loop of the extracellular domain using amino-terminal protein sequencing of purified receptor and by mutagenesis studies. We further show, that a receptor mutant not susceptible to proteolytic cleavage exhibits reduced signaling to G\(_s\) and increased activation of G\(_q\) compared to wild-type PTH1R. These findings indicate that the extracellular domain modulates PTH1R signaling specificity, and that its cleavage affects receptor signaling.}, language = {en} } @article{BoivinBeyersdorfPalmetal.2015, author = {Boivin, Val{\´e}rie and Beyersdorf, Niklas and Palm, Dieter and Nikolaev, Viacheslav O. and Schlipp, Angela and M{\"u}ller, Justus and Schmidt, Doris and Kocoski, Vladimir and Kerkau, Thomas and H{\"u}nig, Thomas and Ertl, Georg and Lohse, Martin J. and Jahns, Roland}, title = {Novel Receptor-Derived Cyclopeptides to Treat Heart Failure Caused by \(Anti-β_1-Adrenoceptor\) Antibodies in a Human-Analogous Rat Model}, series = {PLoS One}, volume = {10}, journal = {PLoS One}, number = {2}, doi = {10.1371/journal.pone.0117589}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-126028}, pages = {e0117589}, year = {2015}, abstract = {Despite recent therapeutic advances the prognosis of heart failure remains poor. Recent research suggests that heart failure is a heterogeneous syndrome and that many patients have stimulating auto-antibodies directed against the second extracellular loop of the \(β_1\) adrenergic receptor \((β_1EC2)\). In a human-analogous rat model such antibodies cause myocyte damage and heart failure. Here we used this model to test a novel antibody-directed strategy aiming to prevent and/or treat antibody-induced cardiomyopathy. To generate heart failure, we immunised n = 76/114 rats with a fusion protein containing the human β1EC2 (amino-acids 195-225) every 4 weeks; n = 38/114 rats were control-injected with 0.9\% NaCl. Intravenous application of a novel cyclic peptide mimicking \(β_1EC2\) (\(β_1EC2-CP\), 1.0 mg/kg every 4 weeks) or administration of the \(β_1-blocker\) bisoprolol (15 mg/kg/day orally) was initiated either 6 weeks (cardiac function still normal, prevention-study, n = 24 (16 treated vs. 8 untreated)) or 8.5 months after the 1st immunisation (onset of cardiomyopathy, therapy-study, n = 52 (40 treated vs. 12 untreated)); n = 8/52 rats from the therapy-study received \(β_1EC2-CP/bisoprolol\) co-treatment. We found that \(β_1EC2-CP\) prevented and (alone or as add-on drug) treated antibody-induced cardiac damage in the rat, and that its efficacy was superior to mono-treatment with bisoprolol, a standard drug in heart failure. While bisoprolol mono-therapy was able to stop disease-progression, \(β_1EC2-CP\) mono-therapy -or as an add-on to bisoprolol- almost fully reversed antibody-induced cardiac damage. The cyclo¬peptide acted both by scavenging free \(anti-β_1EC2-antibodies\) and by targeting \(β_1EC2\)-specific memory B-cells involved in antibody-production. Our model provides the basis for the clinical translation of a novel double-acting therapeutic strategy that scavenges harmful \(anti-β_1EC2-antibodies\) and also selectively depletes memory B-cells involved in the production of such antibodies. Treatment with immuno-modulating cyclopeptides alone or as an add-on to \(β_1\)-blockade represents a promising new therapeutic option in immune-mediated heart failure.}, language = {en} } @article{LohseKlotzSchwabe1991, author = {Lohse, Martin J. and Klotz, Karl-Norbert and Schwabe, Ulrich}, title = {Mechanism of A2 adenosine receptor activation. I. Blockade of A2 adenosine receptors by photoaffinity labeling}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-86073}, year = {1991}, abstract = {It has previously been shown that covalent incorporation of the photoreactive adenosine derivative (R)-2-azido-N6-p-hydroxyphenytisopropyladenosine [(R)-AHPIA] into the A, adenosine receptor of intact fat cells leads to a persistent activation of this receptor, resulting in a reduction of celular cAMP Ieveis [Mol. Pharmacol. 30:403-409 (1986)]. In contrast, covalent incorporation of (R)-AHPIA into human platelet membranes, which contain only stimulatory A2 adenosine receptors, reduces adenytate cyclase Stimulation via these receptors. This effect of (R)-AHPIA is specific for the A2 receptor and can be prevented by the adenosine receptor antagonist theophylline. Binding studies in-dicate that up to 90\% of A2 receptors can be blocked by photoincorporation of (R)-AHPIA. However, the remaining 10-20\% of A2 receptors are sufficient to mediate an adenylate cyclase Stimulation of up to SOOk of the control value. Similarly, the activation via these 10-20\% of receptors occurs with a halflife that is only 2 times Ionger than that in control membranes. This indicates the presence of a receptor reserve, with respect to both the extent and the rate of adenytate cyclase Stimulation. These observations require a modification of the models of receptor-adenytate cyclase coupling, which is described in the accompanying paper [Mol. Pharmacol. 39:524-530 (1991)].}, subject = {Adenosinrezeptor}, language = {en} } @article{GodboleLygaLohseetal.2017, author = {Godbole, Amod and Lyga, Sandra and Lohse, Martin J. and Calebiro, Davide}, title = {Internalized TSH receptors en route to the TGN induce local G\(_{S}\)-protein signaling and gene transcription}, series = {Nature Communications}, volume = {8}, journal = {Nature Communications}, number = {443}, doi = {10.1038/s41467-017-00357-2}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170375}, year = {2017}, abstract = {A new paradigm of G-protein-coupled receptor (GPCR) signaling at intracellular sites has recently emerged, but the underlying mechanisms and functional consequences are insufficiently understood. Here, we show that upon internalization in thyroid cells, endogenous TSH receptors traffic retrogradely to the trans-Golgi network (TGN) and activate endogenous Gs-proteins in the retromer-coated compartment that brings them to the TGN. Receptor internalization is associated with a late cAMP/protein kinase A (PKA) response at the Golgi/TGN. Blocking receptor internalization, inhibiting PKA II/interfering with its Golgi/TGN localization, silencing retromer or disrupting Golgi/TGN organization all impair efficient TSH-dependent cAMP response element binding protein (CREB) phosphorylation. These results suggest that retrograde trafficking to the TGN induces local G\(_{S}\)-protein activation and cAMP/PKA signaling at a critical position near the nucleus, which appears required for efficient CREB phosphorylation and gene transcription. This provides a new mechanism to explain the functional consequences of GPCR signaling at intracellular sites and reveals a critical role for the TGN in GPCR signaling.}, language = {en} } @incollection{LohseKlotzSchwabe1987, author = {Lohse, Martin J. and Klotz, Karl-Norbert and Schwabe, Ulrich}, title = {Functional characterization of A1 adenoosine receptors by photoaffinity labelling}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-86097}, publisher = {Universit{\"a}t W{\"u}rzburg}, year = {1987}, abstract = {The ligand-binding subunit ofthe A1 adenosine receptor has been identified in membranes with the photoaffinity Iabel R-2-azido-N6-p-hydroxyphenylisopropyladenosine (R-AHPIA). Covalent labelling ofthe A1 receptor can also be achieved in intact cells. The dissociation of the radioiodinated label (1251-AHPIA) from isolated rat fat cells was incomplete after UV irradiation, leaving about 20°/o of irreversible specific binding. Such covalent labelling of the receptor led to a concentration-dependent reduction of cellular cyclic AMP levels. This persistent effect of covalent labeHing occurred with an IC50 value of 9 nM, as compared to an IC50 value of 0.9 nM for the direct reduction of cyclic AMP Ievels by the ligand. The difference in the IC5o values can be explained by assuming spare receptors. This hypothesis was verified in binding studies using [ 3HJPIA as a radioligand. R-AHPIA inhibited binding of [3H)PIA to intact fat cells with a K1 value of about 20 nM, which is about 20 tim es high er than the corresponding IC50 value of cyclic AMP reduction. These data show that the A1 receptor is activated according to the occupancy theory. The high sensitivity of the activation in intact ceJis is due to a large number of spare receptors.}, subject = {Adenosinrezeptor}, language = {en} } @article{LohseBockMaiellaroetal.2017, author = {Lohse, Christian and Bock, Andreas and Maiellaro, Isabella and Hannawacker, Annette and Schad, Lothar R. and Lohse, Martin J. and Bauer, Wolfgang R.}, title = {Experimental and mathematical analysis of cAMP nanodomains}, series = {PLoS ONE}, volume = {12}, journal = {PLoS ONE}, number = {4}, doi = {10.1371/journal.pone.0174856}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170972}, pages = {e0174856}, year = {2017}, abstract = {In their role as second messengers, cyclic nucleotides such as cAMP have a variety of intracellular effects. These complex tasks demand a highly organized orchestration of spatially and temporally confined cAMP action which should be best achieved by compartmentalization of the latter. A great body of evidence suggests that cAMP compartments may be established and maintained by cAMP degrading enzymes, e.g. phosphodiesterases (PDEs). However, the molecular and biophysical details of how PDEs can orchestrate cAMP gradients are entirely unclear. In this paper, using fusion proteins of cAMP FRET-sensors and PDEs in living cells, we provide direct experimental evidence that the cAMP concentration in the vicinity of an individual PDE molecule is below the detection limit of our FRET sensors (<100nM). This cAMP gradient persists in crude cytosol preparations. We developed mathematical models based on diffusion-reaction equations which describe the creation of nanocompartments around a single PDE molecule and more complex spatial PDE arrangements. The analytically solvable equations derived here explicitly determine how the capability of a single PDE, or PDE complexes, to create a nanocompartment depend on the cAMP degradation rate, the diffusive mobility of cAMP, and geometrical and topological parameters. We apply these generic models to our experimental data and determine the diffusive mobility and degradation rate of cAMP. The results obtained for these parameters differ by far from data in literature for free soluble cAMP interacting with PDE. Hence, restricted cAMP diffusion in the vincinity of PDE is necessary to create cAMP nanocompartments in cells.}, language = {en} } @article{SchoeneggeGallionPicardetal.2017, author = {Sch{\"o}negge, Anne-Marie and Gallion, Jonathan and Picard, Louis-Philippe and Wilkins, Angela D. and Le Gouill, Christian and Audet, Martin and Stallaert, Wayne and Lohse, Martin J. and Kimmel, Marek and Lichtarge, Olivier and Bouvier, Michel}, title = {Evolutionary action and structural basis of the allosteric switch controlling β\(_2\)AR functional selectivity}, series = {Nature Communications}, volume = {8}, journal = {Nature Communications}, doi = {10.1038/s41467-017-02257-x}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-172268}, year = {2017}, abstract = {Functional selectivity of G-protein-coupled receptors is believed to originate from ligand-specific conformations that activate only subsets of signaling effectors. In this study, to identify molecular motifs playing important roles in transducing ligand binding into distinct signaling responses, we combined in silico evolutionary lineage analysis and structure-guided site-directed mutagenesis with large-scale functional signaling characterization and non-negative matrix factorization clustering of signaling profiles. Clustering based on the signaling profiles of 28 variants of the β\(_2\)-adrenergic receptor reveals three clearly distinct phenotypical clusters, showing selective impairments of either the Gi or βarrestin/endocytosis pathways with no effect on Gs activation. Robustness of the results is confirmed using simulation-based error propagation. The structural changes resulting from functionally biasing mutations centered around the DRY, NPxxY, and PIF motifs, selectively linking these micro-switches to unique signaling profiles. Our data identify different receptor regions that are important for the stabilization of distinct conformations underlying functional selectivity.}, language = {en} }