Regional distribution of the prostaglandin E2 receptor EP1 in the rat brain: accumulation in Purkinje cells of the cerebellum

Prostaglandin E2 (PGE2), is a major prostanoid produced by the activity of cyclooxygenases (COX) in response to various physiological and pathological stimuli. PGE2 exerts its effects by activating four specific E-type prostanoid receptors (EP1, EP2, EP3 and EP4). In the present study, we analyzed the expression of the PGE2 receptor EP1 (mRNA and protein) in different regions of the adult rat brain (hippocampus, hypothalamus, striatum, prefrontal cerebral cortex, parietal cortex, brain stem and cerebellum) using reverse transcription-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemical methods. On a regional basis, levels of EP1 mRNA were the highest in parietal cortex and cerebellum. At the protein level, we found a very strong expression of EP1 in cerebellum as revealed by Western blotting experiments. Furthermore, the present study provides for the first time evidence that the EP1 receptor is highly expressed in the cerebellum, where the Purkinje cells displayed a very high immunolabeling of their perikaryon and dendrites as observed in the immunohistochemical analysis. Results from the present study indicate that the EP1 prostanoid receptor is expressed in specific neuronal populations, which possibly determine the region specific response to PGE2

Analysis of the intracellular transport of misfolded vasopressin V2 receptors

Die Mutationen in den Genen von Membranproteinen fuehren haeufig zu fehlgefalteten Proteinvarianten, die von einem Qualitaetskontrollsystem im fruehen sekretorischen Weg erkannt und retiniert werden. Resultate solcher Mutationen sind oft Ursache fuer vererbbare Krankheiten. Defekte Vasopressin-V2-Rezeptoren (V2R) koennen beispielsweise zu nephrogenem Diabetes Insipidus fuehren. Einen Ansatzpunkt fuer die Behandlung dieser Krankheiten stellen pharmakologische Chaperone dar, die den Faltungsprozess verbessern und dadurch den Transport der Rezeptormutanten zur Zellmembran unterstuetzen. Im Fall des V2R wurde der Antagonist SR121463B als pharmakologisches Chaperon beschrieben. SR121463B bindet mit hoher Affinitaet an den Rezeptor und blockiert die Bindungsstelle des natuerlichen Agonisten 8-Arginin-Vasopressin (AVP). Dadurch wird die Faltung erleichtert und die Rezeptoren koennen besser zur Zellmembran transportiert werden. Aufgrund der hohen Affinitaet von SR121463B und dessen nahezu irreversible Bindung koennen die mutierten Rezeptoren an der Zellmembran nicht mehr durch AVP aktiviert werden. In der vorliegenden Arbeit wurde ein High Throughput Screening (HTS) Verfahren entwickelt, mit dem Ziel, neue Substanzen zu identifizieren, die den Transport von faltungs- und transportdefekten V2R-Mutanten an die Zellmembran verbessern. Mittels dieses HTS wurde eine Bibliothek von ueber 17.500 Substanzen analysiert. Eine identifizierte Hitsubstanz war in der Lage, den Transport der L336T-Mutante des V2R an die Zellmembran stark zu steigern. Die im Rahmen der Entwicklung des HTS-Assays etablierte automatische Mikroskopie wurde auch dazu genutzt, Transportstudien an anderen G-Protein-gekoppelten Rezeptoren durchzufuehren, beispielsweise an Corticotropin-Releasing-Factor- Rezeptoren. Bisher war ungeklaert, ob pharmakologische Chaperone co- und/oder post-translational wirken. Um dieses zu untersuchen, wurden im zweiten Teil der Arbeit Fusionen des wildtypischen V2R und der fehlgefalteten Rezeptormutanten S167T und R337X mit dem photokonvertierbaren Fluoreszenzprotein mKikGR hergestellt. Mit Hilfe eines neu etablierten zellbasierten Assays wurde nachgewiesen, dass SR121463B sowohl co- als auch post-translational wirken kann.Mutations in the genes encoding for membrane proteins lead to misfolded protein forms that are recognized by a quality control system in the early secretory pathway. Misfolded proteins are usually retained intracellularly and may cause inherited diseases. In the case of the vasopressin V2 receptor (V2R) mutations may cause nephrogenic diabetes insipidus. A pharmacological approach to treat diseases where misfolded proteins play a role involves the use of pharmacological chaperones, which bind specifically to the native proteins, improve protein folding and consequently rescue the transport of the mutant receptors to the cell membrane. For the V2R, the antagonist SR121463B has been described as a pharmacological chaperone. SR121463B binds with very high affinity (i.e. almost irreversibly) to the receptor, with the consequence that the natural ligand 8-arginin-vasopressin (AVP) can not activate rescued receptors because the ligand binding pocket is blocked. In the present study, a high throughput screening assay (HTS) based on automated microscopy was developed in order to identify novel compounds which are able to rescue the trafficking of misfolded and transport defective V2R-mutants to the cell membrane. Using this HTS assay, a library containing over 17,500 compounds was analysed. One hit compound was found which was able to rescue significantly the transport of the L336T mutant of the V2R to the cell membrane. In various cooperations, the HTS assay was also used to analyze trafficking of other GPCRs such as corticotropin releasing factor receptors. To date, it was not completely clear whether pharmacological chaperones act co or post- translationally. In order to address this question, fusions of the wild type V2R and its misfolded receptor mutants S167T and R337X were generated with the photoconvertible fluorescent protein mKikGR in the second part of this study. By using a novel cell-based assay using mKikGR, it could be shown that the pharmacological chaperone SR1214363B can act co- as well as post- translationally

Inhibition of Biosynthesis of Human Endothelin B Receptor by the Cyclodepsipeptide Cotransin

The specific inhibition of the biosynthesis of target proteins is a relatively novel strategy in pharmacology and is based mainly on antisense approaches (e.g. antisense oligonucleotides or RNA interference). Recently, a novel class of substances was described acting at a later step of protein biosynthesis. The cyclic heptadepsipeptides CAM741 and cotransin were shown to inhibit selectively the biosynthesis of a small subset of secretory proteins by preventing stable insertion of the nascent chains into the Sec61 translocon complex at the endoplasmic reticulum membrane (Besemer, J., Harant, H., Wang, S., Oberhauser, B., Marquardt, K., Foster, C. A., Schreiner, E. P., de Vries, J. E., Dascher-Nadel, C., and Lindley, I. J. (2005) Nature 436, 290–293; Garrison, J. L., Kunkel, E. J., Hegde, R. S., and Taunton, J. (2005) Nature 436, 285–289). These peptides act in a signal sequence-discriminatory manner, which explains their selectivity. Here, we have analyzed the cotransin sensitivity of various G protein-coupled receptors in transfected HEK 293 cells. We show that the biosynthesis of the human endothelin B receptor (ETBR) is highly sensitive to cotransin, in contrast to that of the other G protein-coupled receptors analyzed. Using a novel biosynthesis assay based on fusions with the photoconvertible Kaede protein, we show that the IC50 value of cotransin action on ETBR biosynthesis is 5.4 μM and that ETBR signaling could be completely blocked by treating cells with 30 μM cotransin. Taken together, our data add an integral membrane protein, namely the ETBR, to the small group of cotransin-sensitive proteins