Rainer Strotmann's research while affiliated with University of Leipzig and other places
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Publications (49)
RGS9-deficient mice show drug-induced dyskinesia but normal locomotor activity under unchallenged conditions.
Genes related to Ca2+ signaling and their functions were regulated in RGS9-deficient mice.
Changes in Ca2+ signaling that compensate for RGS9 loss-of-function can explain the normal locomotor activity in RGS9-deficient mice under unchalleng...
Interaction and cross-talk of G-protein-coupled receptors (GPCRs) are of considerable interest because an increasing number
of examples implicate a profound functional and physiological relevance of homo- or hetero-oligomeric GPCRs. The ghrelin (growth
hormone secretagogue receptor (GHSR)) and melanocortin-3 (MC3R) receptors are both known to have...
Interaction and cross-talk of G-protein-coupled receptors (GPCRs) are of considerable interest because an increasing number of examples implicate a profound functional and physiological relevance of homo- or hetero-oligomeric GPCRs. The ghrelin (growth hormone secretagogue receptor (GHSR)) and melanocortin-3 (MC3R) receptors are both known to have...
Once introduced into the very early eukaryotic blueprint, seven-transmembrane receptors soon became the central and versatile components of the evolutionary highly successful G protein-coupled transmembrane signaling mechanism. In contrast to all other components of this signal transduction pathway, G protein-coupled receptors (GPCR) evolved in var...
Identification of CaM binding and interdomain interaction sites in TRPV4. (A) A library of 20-mer peptides overlapping by 18 amino acids and covering the TRPV4 N terminus was spotted on filter paper and probed with biotinylated CaM or C2 at 100 µM Ca2+. The peptide dots are shown at the start of the respective 20-mer peptide within the TRPV4 sequen...
Ca2+-dependent binding of C2 to CaM or CaM mutants that are partially Ca2+-binding defective. The Ca2+-dependence of C2-biotin binding to wild type CaM-Flag or mutants that are Ca2+-binding deficient in the N lobe (CaM12), the C lobe (CaM34) or both lobes (CaM1234) was measured in an AlphaScreen assay at the indicated Ca2+ concentrations. Half maxi...
The N2 fragment forms dimers in gel filtration experiments. (A) N2 results in a monophasic elution profile with an apparent molecular size that corresponds with a dimeric form of the protein. (B) Calibration was done with a commercial protein mixture with the indicated molecular weights.
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CaM binding properties of the putative CaM interaction peptides.
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CaM binding in the CaM interaction sites in TRPV4. (A–E), In fluorescence polarization experiments, the carboxyfluorescein-labeled peptides P1 (A), P2 (B), P3 (C), P4 (D) and P5 (E) were incubated with CaM, CaM mutants or isolated CaM lobes (see Fig. 1B in main text) at concentrations between 0.1 nM and 10 µM. The respective EC50 values are given i...
Several Ca(2+)-permeable channels, including the non-selective cation channel TRPV4, are subject to Ca(2+)-dependent facilitation. Although it has been clearly demonstrated in functional experiments that calmodulin (CaM) binding to intracellular domains of TRP channels is involved in this process, the molecular mechanism remains elusive. In this st...
Positions of the TRPV4 fragments used in the study.
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Supplementary Methods.
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AlphaScreen-based CaM interaction assay. Streptavidin-coated AlphaScreen donor- and anti-Flag-coated acceptor beads were incubated with the indicated concentrations of CaM-biotin and 6 nM C2-Flag (A) or C2-Flag and 10 nM CaM-biotin (B). Data points show mean±SEM of measurements in buffer containing 100 µM Ca2+ (filled squares) or 1 mM EGTA (open sq...
Controlling enzyme activity by ligand binding to a regulatory domain of choice may have many applications e.g. as biosensors and as tools in regulating cellular functions. However, until now only a small number of ligand-binding domains have been successfully linked to enzyme activity. G protein-coupled receptors (GPCR) are capable of recognizing a...
Modulation of enzyme activity in M3R-luciferase fusion proteins by different receptor ligands. COS-7 cells transfected with M3R-luci and construct #8 were stimulated with the indicated ligands (100 μM) and luciferase activity was determined. The luminescence of the fusion constructs were 184,314 ± 75,049 AU (M3R-luci) and 140,452 ± 55,426 AU (const...
M3R ligands have no effect on soluble luciferase and V2R-luciferase fusion protein. COS-7 cells were transfected with luciferase (A) and V2R-luci (B). Increasing concentrations of CCh (square), atropine (open circle), scopolamine (filled circle) and butylscopolamine (diamond) were applied and luciferase activity was determined as described under Me...
Kinetics of luciferase activity assay. Cells were transfected with M3R-luci and luciferase and the assay was performed as described in the Method section with the exception that the luciferase buffer was added at different incubation times after lysis. Data are given as mean ± S.D. of one experiment performed in triplicate.
Luciferase can be integrated into other GPCR (e.g. V2R) without loosing G protein-coupling abilities. COS-7 cells were transfected with wild type V2R and V2R-luci. 48 h after transfection cells were incubated with 100 nM arginine-vasopressin (AVP). Cyclic AMP levels were determined using the non-radioactive cAMP-determination kit (AlphaScreening te...
Description of control experiments. This file summarises all control experiments carried out.
Primer used in this study. This table listes all primers used to generate the mutants of the M3R-luciferase fusion protein.
Polyuria, hypernatremia, and hypovolemia are the major clinical signs of inherited nephrogenic diabetes insipidus (NDI). Hypernatremia is commonly considered a secondary sign caused by the net loss of water due to insufficient insertion of aquaporin-2 water channels into the apical membrane of the collecting duct cells. In the present study, we emp...
The protease cathepsin D (Cath D) and its proteolytically inactive proform, procathepsin D (ProCath D), turned out to be multifunctional within and outside the cell. Elevated levels of ProCath D occur in malignant tumors and in organs under chronic inflammation. One important source for this increase of ProCath D might be endothelial cells. Here we...
Supplementary Results and Discussion, and Materials and Methods
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Table of PCR and sequencing primers
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Visual haplotype graph and network of the SeattleSNPs sequence data for the TRPV6 locus. A) A visual haplotype graph of the TRPV6 locus. Each horizontal line is an individual haplotype from a given individual, and each vertical column is a SNP marked by the position of that SNP in the sequence. The major alleles are in blue, while minor alleles are...
SeattleSNPs Genome Scan Statistics
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Summary Statistics for EPHB6, TRPV6, TRPV5 and KEL
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A scatter plot of molecular Fst and lnRHap values for each of the 221 genes from the SeattleSNPs data set. Genes from the candidate region of chromosome 7q34, including the TRPV6 locus, are identified in the plot.
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PCR primers for MALDI-TOF assay
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A topology graph of the TRPV6 protein illustrating the location of the three non-synonymous mutations, C157R, M378R and M681T and their relationship with putative important functional areas.
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Visual haplotype graph of the TRPV5 locus from the SeattleSNPs data set. Each horizontal line is a haplotype and each vertical column is a SNP marked by its position in the TRPV5 sequence. Major alleles are marked in blue, and minor alleles are in yellow. All genotypes were used to infer the haplotypes.
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An overview of the genomic region encompassing the TRPV6/TRPV5 region used to sequence eight blocks in the Karitiana, Han Chinese, highland Papua New Guineans, and the Pathan, previously sequenced in Europeans and African-Americans. The illustration is from the UCSC Genome Browser (chr7:142,278,427-142,330,351) and with the inclusion of a custom tr...
Haplotype frequencies of three non-synonymous polymorphisms used as tagging SNPs (rs4987657 (C157R), rs4987667 (M378V) and rs4987682 (M681T)) and genotyped in the CEPH Human Diversity panel and additionally in 31 Ethiopians, 51 Germans, 11 South African Bantu-speakers, and three hunter-gatherer groups from India (44 Koragas, 16 Mullukurunan, and 2...
A density plot of the time since fixation for the putative allele in Europeans, with estimations are based on twelve and ten segregating sites. The 2.5% and 97.5% credible intervals are plotted for both time estimations. The average mode values is ∼7,000 ybp.
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World Haplotype Frequencies for C157R, M378V and M681T
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We identified and examined a candidate gene for local directional selection in Europeans, TRPV6, and conclude that selection has acted on standing genetic variation at this locus, creating parallel soft sweep events in humans. A novel modification of the extended haplotype homozygosity (EHH) test was utilized, which compares EHH for a single allele...
Genome-wide scans for positive selection in humans provide a promising approach to establish links between genetic variants and adaptive phenotypes. From this approach, lists of hundreds of candidate genomic regions for positive selection have been assembled. These candidate regions are expected to contain variants that contribute to adaptive pheno...
The range of actions of the second messenger Ca(2+) is a key determinant of neuronal excitability and plasticity. For dendritic spines, there is on-going debate regarding how diffusional efflux of Ca(2+) affects spine signalling. However, the consequences of spino-dendritic coupling for dendritic Ca(2+) homeostasis and downstream signalling cascade...
TRPV4 is a non-selective cation channel subunit expressed in a wide variety of tissues. TRP channels are formed by a tetrameric complex of channel subunits. The available evidence suggests that TRPV4 cannot form heteromultimers with other TRPV isoforms, and that TRPV4-containing channels are homotetramers. These channels have a characteristic outwa...
Mammalian TRP channels form a large family with around thirty members. From sequence similarity, TRPs can be divided into three major TRP subfamilies: the classical or canonical subfamily (TRPC), the melastatin-related subfamily (TRPM), and the vanilloid-receptor–related subfamily (TRPV) [1]. In addition, there are a number of more distantly relate...
Maintenance of water and electrolyte homeostasis is central to mammalian survival and, therefore, under stringent hormonal control. Water homeostasis is achieved by balancing fluid intake with water excretion, governed by the antidiuretic action of arginine vasopressin. Arginine vasopressin stimulation of renal V2 vasopressin receptors in the basol...
Most Ca2+-permeable ion channels are inhibited by increases in the intracellular Ca2+ concentration ([Ca2+]i), thus preventing potentially deleterious rises in [Ca2+]i. In this study, we demonstrate that currents through the osmo-, heat- and phorbol ester-sensitive, Ca2+-permeable nonselective cation channel TRPV4 are potentiated by intracellular C...
Mammalian members of the classical transient receptor potential channel (TRPC) subfamily (TRPC1-7) are Ca(2+)-permeable cation channels involved in receptor-mediated increases in intracellular Ca(2+). Unlike most other TRP-related channels, which are inhibited by La(3+) and Gd(3+), currents through TRPC4 and TRPC5 are potentiated by La(3+). Because...
To investigate the possible role of members of the mammalian transient receptor potential (TRP) channel family (TRPC1-7) in vasoconstrictor-induced Ca(2+) entry in vascular smooth muscle cells, we studied [Arg(8)]-vasopressin (AVP)-activated channels in A7r5 aortic smooth muscle cells. AVP induced an increase in free cytosolic Ca(2+) concentration...
Ca2+-permeable channels that are involved in the responses
of mammalian cells to changes in extracellular osmolarity have not been characterized
at the molecular level. Here we identify a new TRP (transient receptor potential)-like
channel protein, OTRPC4, that is expressed at high levels in the kidney, liver
and heart. OTRPC4 forms Ca2+-permeable,...
Citations
... Mouse basal ganglia (MBG) tissue samples were used as a positive control alongside Area X. According to the product sheet and in mouse literature, the band observed around 55 kD is CaMKIIB and the band detected around 48 kD is the IgG heavy chain [26]. The IgG heavy chain band was not included for analysis. ...
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... Effect of heterodimerization on GHSR1a and GPCRs functions[101][102][103][104][105][106][107][108][109]. ...
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... Over the last years, the ghrelin receptor (GHS-R1a) has been shown to heterodimerize with several other GPCRs, including the neurotensin receptor 1 (NTS1R; Takahashi et al., 2006), the dopamine D1R (Jiang et al., 2006;Schellekens et al., 2013a) and D2R (Kern et al., 2012); the melanocortin receptor 3 (MCR3; Rediger et al., 2011;Muller et al., 2013;Schellekens et al., 2013a) and the 5HT2CR (Schellekens et al., 2013a;Kern et al., 2015). For reviews on ghrelin and its receptor forming the mentioned heterocomplexes, see Schellekens et al. (2013b), Borroto-Escuela et al. (2014), Wellman and Abizaid (2015), and Ringuet et al. (2021). ...
... TRPV4 is the fourth cloned member of the TRPV subfamily reported as a modulator of uterine contractility in rats [7,8]. TRPV4 can be activated by diverse stimuli, including osmotic or mechanical stimuli, warm temperature, phorbol ester derivatives, products of arachidonic acid breakdown or feedback regulation by Ca 2+ [9]. As the prominent co-localization of TRPV4 and AQP4 in retinal Müller glia cells was earlier identified Life 2021, 11, 897 2 of 9 by Ryskamp DA et al. [10], and we also determined the co-expression of TRPV4 and AQP5 in late-pregnant rat uterine tissue [7]. ...
... This classification system was mainly designed to cover all vertebrate and invertebrate GPCRs. Although the sequence similarities among GPCRs have largely diverged, previous studies suggested that this superfamily may have originated and diversified in early eukaryotic evolution [4,5]. Due to their important roles, GPCRs have been widely identified and studied in many species, especially in model organisms. ...
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... These findings suggested that Ca 2+ -dependent inhibition of TRPV4 channel does not rely on CCaM binding to CCaMB. Strotmann and colleagues (Strotmann et al., 2010) proposed a mechanism whereby an interaction between the N-and C-terminal tails prevents channel activation. An increase in cytosolic Ca 2+ enabled CaM binding to the CCaMB, which resulted in the displacement of N-terminal from the C-terminal tail and TRPV4 channel activation. ...
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... Thereafter, cells were washed once with serum-free DMEM containing 10 mM LiCl followed by incubation with serumfree DMEM containing 10 mM LiCl. Empty vector (eV) transfected cells stimulated with 10 mM of forskolin and the M3 muscarinic acetylcholine receptor (M3R) [29] transfected cells stimulated with 10 mM of carbachol, for 1 h at 37 C served as positive controls for the cAMP and IP assays, respectively. Intracellular IP levels were determined by anion-exchange chromatography as previously described [30]. ...
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... These findings indicate that FIBIN induces the expression of Tbx5, although the signaling cascade is still unknown. FIBIN mRNA has also been found to be expressed at increased levels of neonatal mice born with X-linked nephrogenic diabetes insipidus (Schliebe et al., 2008). qPCR studies of different stages of mouse embryos found that expression of FIBIN was highest in the cerebellum, skeletal muscle, and trunk of embryos at 13-16 days post fertilization (Lakner et al., 2011). ...
... The experiments were performed on cloned full-length human TRPV1 (Caterina et al., 1997), TRPV3 (Peier et al., 2002;Smith et al., 2002), and TRPV4 (Liedtke et al., 2000;Strotmann et al., 2000) channels in pV10 mammalian expression vector with a C-terminal EGFP tag. ...
... The data represent the mean values ± SEM of n number of animals. (c) Representative trace recording of schwarzinicine A in rat isolated aorta Schultz, & Plant, 2002). Interestingly, the expression of TRPC channels was found to increase in VSMCs isolated from hypertensive animals and patients (Chen et al., 2010;Dietrich et al., 2005). ...
