@article{BoeerNeuschaeferRubeMoelleretal.2000, author = {B{\"o}er, Ulrike and Neusch{\"a}fer-Rube, Frank and M{\"o}ller, Ulrike and P{\"u}schel, Gerhard Paul}, title = {Requirement of N-glycosylation of the prostaglandin E2 receptor EP3beta for correct sorting to the plasma membrane but not for correct folding}, year = {2000}, abstract = {Eight heptahelical receptors have been characterized for prostaglandin (PG) D(2), PGE(2), PGF(2alpha), prostacyclin and thromboxane A(2). They share a sequence identity of 40\%. All of them have potential N-glycosylation sites. The current study analysed the role of the two N-glycosylation sites in the rat EP3beta-subtype PGE(2) receptor for protein folding and sorting. The N-glycosylation consensus sequences were eliminated by site-directed mutagenesis and receptors expressed in HEK-293 cells. Both potential N-glycosylation sites were used. Their joint elimination resulted in the synthesis of a receptor protein with full binding competence, biological activity and no reduction of affinity; however, the half-life of the non-glycosylated receptor was slightly reduced. Ligand binding to intact stably transfected cells and confocal laser microscopic immunocytochemistry showed that the glycosylated receptor was correctly inserted into the plasma membrane to a much larger extent than the non-glycosylated receptor, which tended to accumulate in the perinuclear zone of the endoplasmic reticulum. Inhibition of N-glycosylation with tunicamycin resulted in a similar perinuclear distribution of the wild-type receptor. Therefore, glycosylation of the EP3beta receptor seems not to be necessary for correct folding of the receptor protein but for the efficient transport of the receptor protein to the plasma membrane. This contrasts with a previous finding which described a reduction of the affinity for PGE(2) of the EP3alpha receptor by elimination of the distal glycosylation site when the receptor protein was expressed in insect cells.}, language = {en} } @article{GiebingToelleJuergensenetal.2005, author = {Giebing, G{\"u}nter and T{\"o}lle, Markus and J{\"u}rgensen, Jana and Eichhorst, Jenny and Furkert, Jens and Beyermann, Michael and Neusch{\"a}fer-Rube, Frank and Rosenthal, Walter and Zidek, Walter and van der Giet, Markus and Oksche, Alexander}, title = {Arrestin-independent internalization and recycling of the urotensin receptor contribute to long-lasting urotensin II - Mediated vasoconstriction}, issn = {0009-7330}, year = {2005}, abstract = {Urotensin II (UII), which acts on the G protein-coupled urotensin ( UT) receptor, elicits long-lasting vasoconstriction. The role of UT receptor internalization and intracellular trafficking in vasoconstriction has yet not been analyzed. Therefore, UII-mediated contractile responses of aortic ring preparations in wire myography and rat UT (rUT) receptor internalization and intracellular trafficking in binding and imaging analyses were compared. UII elicited a concentration-dependent vasoconstriction of rat aorta (-log EC50, mol/L:9.0 +/- 0.1). A second application of UII after 30 minutes elicited a reduced contraction (36 +/- 4\% of the initial response), but when applied after 60 minutes elicited a full contraction. In internalization experiments with radioactive labeled VII (I-125-UII), approximate to 70\% of rUT receptors expressed on the cell surface of human embryonic kidney 293 cells were sequestered within 30 minutes (half life [t(h)]: 5.6 +/- 0.2 minutes), but recycled quantitatively within 60 minutes (t(h) 31.9 +/- 2.6 minutes). UII- bound rUT receptors were sorted to early and recycling endosomes, as evidenced by colocalization of rUT receptors with the early endosomal antigen and the transferrin receptor. Real-time imaging with a newly developed fluorescent UII (Cy3- UII) revealed that rUT receptors recruited arrestin3 green fluorescent protein to the plasma membrane. Arrestin3 was not required for the endocytosis of the rUT receptor, however, as internalization of Cy3-UII was not altered in mouse embryonic fibroblasts lacking endogenous arrestin2/arrestin3 expression. The data demonstrate that the rUT receptor internalizes arrestin independently and recycles quantitatively. The continuous externalization of rUT receptors provides the basis for repetitive and lasting UII-mediated vasoconstriction}, language = {en} } @phdthesis{NeuschaeferRube2003, author = {Neusch{\"a}fer-Rube, Frank}, title = {Struktur-Funktionsbeziehungen von Postaglandinrezeptoren sowie transkriptionelle und posttranslationale Regulation ihrer Aktivit{\"a}t}, pages = {Getr. Z{\"a}hlung}, year = {2003}, language = {de} } @misc{NeuschaeferRubeDeVriesHaeneckeetal.1994, author = {Neusch{\"a}fer-Rube, Frank and DeVries, Christa and H{\"a}necke, Kristina and Jungermann, Kurt and P{\"u}schel, Gerhard Paul}, title = {Molecular cloning and expression of a prostaglandin E₂ receptor of the EP₃ϐ subtype from rat hepatocytes}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-45830}, year = {1994}, abstract = {Rat hepatocytes have previously been reported to possess prostaglandin E₂ receptors of the EP₃-type (EP₃-receptors) that inhibit glucagonstimulated glycogenolysis by decreasing cAMP. Here, the isolation of a functional EP₃ϐ receptor cDNA clone from a rat hepatocyte cDNA library is reported. This clone can be translated into a 362-amino-acid protein, that displays over 95\% homology to the EP₃ϐ receptor from mouse mastocytoma. The amino- and carboxy-terminal region of the protein are least conserved. Transiently transfected HEK 293 cells expressed a single binding site for PGE₂ with an apparent Kd of 15 nM. PGE₂ > PGF₂α > PGD₂ competed for [³H]PGE₂ binding sites as did the EP₃ receptor agonists M\&B 28767 = sulprostone > misoprostol but not the EP₁ receptor antagonist SC 19220. In stably transfected CHO cells M\&B 28767 > sulprostone = PGE₂ > misoprostol > PGF₂α inhibited the forskolin-elicited cAMP formation. Thus, the characteristics of the EP₃ϐ receptor of rat hepatocytes closely resemble those of the EP₃ϐ receptor of mouse mastocytoma.}, language = {en} } @article{NeuschaeferRubeEngemaierKochetal.2003, author = {Neusch{\"a}fer-Rube, Frank and Engemaier, Eva and Koch, Sina and B{\"o}er, Ulrike and P{\"u}schel, Gerhard Paul}, title = {Identification by site-directed mutagenesis of amino acids contributing to ligand-binding specificity or signal transduction properties of the human FP prostanoid receptor}, year = {2003}, abstract = {Prostanoid receptors belong to the class of heptahelical plasma membrane receptors. For the five prostanoids, eight receptor subtypes have been identified. They display an overall sequence similarity of roughly 30\%. Based on sequence comparison, single amino acids in different subtypes of different species have previously been identified by site-directed mutagenesis or in hybrid receptors that appear to be essential for ligand binding or G-protein coupling. Based on this information, a series of mutants of the human FP receptor was generated and characterized in ligand- binding and second-messenger-formation studies. It was found that mutation of His-81 to Ala in transmembrane domain 2 and of Arg-291 to Leu in transmembrane domain 7, which are putative interaction partners for the prostanoid's carboxyl group, abolished ligand binding. Mutants in which Ser-263 in transmembrane domain 6 or Asp-300 in transmembrane domain 7 had been replaced by Ala or Gln, respectively, no longer discriminated between prostaglandins PGF(2alpha) and PGD(2). Thus distortion of the topology of transmembrane domains 6 and 7 appears to interfere with the cyclopentane ring selectivity of the receptor. PGF(2alpha)-induced inositol formation was strongly reduced in the mutant Asp-300Gln, inferring a role for this residue in agonist-induced G-protein activation.}, language = {en} } @article{NeuschaeferRubeHermosillaKunaetal.2004, author = {Neusch{\"a}fer-Rube, Frank and Hermosilla, Ricardo and Kuna, Manuela and Pathe-Neuschaefer-Rube, Andrea and P{\"u}schel, Gerhard Paul}, title = {Agonist-induced desensitization of rat prostaglandin EP3 receptor isoforms}, issn = {0028-1298}, year = {2004}, language = {en} } @article{NeuschaeferRubeHermosillaKunaetal.2005, author = {Neusch{\"a}fer-Rube, Frank and Hermosilla, Ricardo and Kuna, Manuela and Pathe-Neusch{\"a}fer-Rube, Andrea and Schulein, R. and P{\"u}schel, Gerhard Paul}, title = {A Ser/Thr cluster within the C-terminal domain of the rat prostaglandin receptor EP3 alpha is essential for agonist-induced phosphorylation, desensitization and internalization}, issn = {0007-1188}, year = {2005}, abstract = {1 Two isoforms of the rat prostaglandin E-2 receptor, rEP3 alpha-R and rEP3 beta-R, differ only in their C- terminal domain. To analyze the function of the rEP3-R C-terminal domain in agonist induced desensitization, a cluster of Ser/Thr residues in the C-terminal domain of the rEP3 alpha-R was mutated to Ala and both isoforms and the receptor mutant (rEP3 alpha-ST341-349A-R) were stably expressed in HEK293 cells. 2 All rEP3-R receptors showed a similar ligand- binding profile. They were functionally coupled to Gi and reduced forskolin-induced cAMP-formation. 3 Repeated exposure of cells expressing the rEP3 alpha-R isoform to PGE(2) reduced the agonist induced inhibition of forskolin-stimulated cAMP-formation by 50\% and led to internalization of the receptor to intracellular endocytotic vesicles. By contrast, Gi- response as well as plasma membrane localization of the rEP3 beta-R and the rEP3 alpha-ST341-349A-R were not affected by prior agonist-stimulation. 4 Agonist-stimulation of HEK293-rEP3 alpha-R cells induced a time- and dose-dependent phosphorylation of the receptor most likely by G protein-coupled receptor kinases and not by protein kinase A or protein kinase C. By contrast, upon agonist-stimulation the rEP3 beta-R was not phosphorylated and the rEP3 alpha-ST341-349A-R was phosphorylated only weakly. 5 These results led to the hypothesis that agonist-induced desensitization of the rEP3 alpha-R isoform is mediated most likely by a GRK-dependent phosphorylation of Ser/Thr residues 341 - 349. Phosphorylation then initiates uncoupling of the receptor from Gi protein and receptor internalization}, language = {en} } @article{NeuschaeferRubeHermosillaRehwaldetal.2004, author = {Neusch{\"a}fer-Rube, Frank and Hermosilla, Ricardo and Rehwald, Matthias and Ronnstrand, Lars and Sch{\"u}lein, Ralf and Wernstedt, Christer and P{\"u}schel, Gerhard Paul}, title = {Identification of a Ser/Thr cluster in the C-terminal domain of the human prostaglandin receptor EP4 that is essential for agonist-induced beta-arrestin1 recruitment but differs from the apparent principal phosphorylation site}, year = {2004}, abstract = {hEP4-R (human prostaglandin E2 receptor, subtype EP4) is a G(s)-linked heterotrimeric GPCR (G-protein-coupled receptor). It undergoes agonist-induced desensitization and internalization that depend on the presence of its C- terminal domain. Desensitization and internalization of GPCRs are often linked to agonist-induced beta-arrestin complex formation, which is stabilized by phosphorylation. Subsequently beta-arrestin uncouples the receptor from its G-protein and links it to the endocytotic machinery. The C-terminal domain of hEP4-R contains 38 Ser/Thr residues that represent potential phosphorylation sites. The present study aimed to analyse the relevance of these Ser/Thr residues for agonist- induced phosphorylation, interaction with beta-arrestin and internalization. In response to agonist treatment, hEP4-R was phosphorylated. By analysis of proteolytic phosphopeptides of the wild-type receptor and mutants in which groups of Ser/Thr residues had been replaced by Ala, the principal phosphorylation site was mapped to a Ser/Thr-containing region comprising residues 370-382, the presence of which was necessary and sufficient to obtain full agonist-induced phosphorylation. A cluster of Ser/Thr residues (Ser-389-Ser-390-Thr-391-Ser-392) distal to this site, but not the principal phosphorylation site, was essential to allow agonist-induced recruitment of beta-arrestin1. However, phosphorylation greatly enhanced the stability of the beta-arrestin1-receptor complexes. For maximal agonist-induced internalization, phosphorylation of the principal phosphorylation site was not required, but both beta-arrestin1 recruitment and the presence of Ser/Thr residues in the distal half of the C-terminal domain were necessary.}, language = {en} } @article{NeuschaeferRubeMoellerPueschel2000, author = {Neusch{\"a}fer-Rube, Frank and M{\"o}ller, Ulrike and P{\"u}schel, Gerhard Paul}, title = {Structure of the 5'-flanking region of the rat prostaglandin f(2alpha) receptor}, year = {2000}, abstract = {Prostaglandin F(2alpha) (PGF(2alpha)), modulates hepatocyte functions via a heptahelical G(q)-coupled PGF(2alpha)-receptor (FP-R) which in liver is expressed exclusively in hepatocytes. The aim of the present study was to isolate the 5'-flanking region of the rat FP-R gene and to elucidate its basal and IL-6-modulated transcription control function in rat hepatocytes. The 5'-non-translated region of the rat hepatocyte FP-R mRNA differed from the corresponding region in rat fetal astrocyte or corpus luteum. It was encoded by exons 1a and 2 which were separated by a 1. 4 kb intron containing the exons 1b and 1c coding for the 5'-untranslated region of rat fetal astrocyte and corpus luteum FP-R mRNA, respectively. The transcription initiation site in hepatocytes was localized 263 bp upstream of the start ATG by 5'-RACE. A DNA-fragment covering the 5'-flanking region of the rFP-R gene from - 1 of the transcription initiation site to -2590 bp was cloned and sequenced. Its 3'-two thirds had a 65\% sequence identity to the mouse FP-R promoter however no homology to the bovine FP-R promoter. In the overlapping sequence most of the putative transcription factor binding sites were conserved between mouse and rat. The rat promoter contained no classical TATA- or CAAT-boxes but putative binding sites for the transcription factors C/EBP, GATA-1, HNF-1, HNF-3beta, SP-1, and USF. Luciferase reporter gene constructs containing portions of the 5'-flanking region were transfected into rat hepatocytes. Luciferase expression ranked -181 >/= -608 < -1418 > -1821 >/= -2590. The strongest transcriptional activity was conferred by the region between -608 and -1418 containing a cluster of potential HNF-1 and HNF-3beta binding sites that might allow the exclusive expression of FP-R mRNA in hepatocytes. The amount of FP-R mRNA and the luciferase expression under control of the -2590 promoter fragment were reduced by IL-6 in hepatocytes. Copyright 2000 Academic Press.}, language = {en} } @article{NeuschaeferRubeOppermannMoelleretal.1999, author = {Neusch{\"a}fer-Rube, Frank and Oppermann, Martin and M{\"o}ller, Ulrike and B{\"o}er, Ulrike and P{\"u}schel, Gerhard Paul}, title = {Agonist-induced phosphorylation by G protein-coupled receptor kinases of the EP4 receptor carboxyl-terminal domain in an EP3/EP4 prostaglandin E(2) receptor hybrid}, issn = {1521-0111}, year = {1999}, abstract = {Prostaglandin E(2) receptors (EP-Rs) belong to the family of heterotrimeric G protein-coupled ectoreceptors with seven transmembrane domains. They can be subdivided into four subtypes according to their ligand-binding and G protein-coupling specificity: EP1 couple to G(q), EP2 and EP4 to G(s), and EP3 to G(i). The EP4-R, in contrast to the EP3beta-R, shows rapid agonist-induced desensitization. The agonist-induced desensitization depends on the presence of the EP4-R carboxyl-terminal domain, which also confers desensitization in a G(i)-coupled rEP3hEP4 carboxyl-terminal domain receptor hybrid (rEP3hEP4-Ct-R). To elucidate the possible mechanism of this desensitization, in vivo phosphorylation stimulated by activators of second messenger kinases, by prostaglandin E(2), or by the EP3-R agonist M\&B28767 was investigated in COS-7 cells expressing FLAG-epitope-tagged rat EP3beta-R (rEP3beta-R), hEP4-R, or rEP3hEP4- Ct-R. Stimulation of protein kinase C with phorbol-12-myristate-13-acetate led to a slight phosphorylation of the FLAG- rEP3beta-R but to a strong phosphorylation of the FLAG-hEP4-R and the FLAG-rEP3hEP4-Ct-R, which was suppressed by the protein kinase A and protein kinase C inhibitor staurosporine. Prostaglandin E(2) stimulated phosphorylation of the FLAG- hEP4-R in its carboxyl-terminal receptor domain. The EP3-R agonist M\&B28767 induced a time- and dose-dependent phosphorylation of the FLAG-rEP3hEP4-Ct-R but not of the FLAG-rEP3beta-R. Agonist-induced phosphorylation of the FLAG- hEP4-R and the FLAG-rEP3hEP4-Ct-R were not inhibited by staurosporine, which implies a role of G protein-coupled receptor kinases (GRKs) in agonist-induced receptor phosphorylation. Overexpression of GRKs in FLAG-rEP3hEP4-Ct-R- expressing COS-7 cells augmented the M\&B28767-induced receptor phosphorylation and receptor sequestration. These findings indicate that phosphorylation of the carboxyl-terminal hEP4-R domain possibly by GRKs but not by second messenger kinases may be involved in rapid agonist-induced desensitization of the hEP4-R and the rEP3hEP4-Ct-R.}, language = {en} }