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Institute
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
1) During orthograde perfusion of rat liver human anaphylatoxin C3a caused an increase in glucose and lactate output and reduction of flow. These effects could be enhanced nearly twofold by co-infusion of the carboxypeptidase inhibitor MERGETPA, which reduced inactivation of C3a to C3adesArg. 2) During retrograde perfusion C3a caused a two- to threefold larger increase in glucose and lactate output and reduction of flow than in orthograde perfusions. These actions tended to be slightly enhanced by MERGETPA. 3) The elimination of C3a plus C3adesArg immunoreactivity during a single liver passage was around 67%, irrespective of the perfusion direction and the presence of the carboxypeptidase inhibitor MERGETPA; however, less C3adesArg and more intact C3a appeared in the perfusate in the presence of MERGETPA in orthograde and retrogade perfusions It is concluded that rat liver inactivated human anaphylatoxin C3a by conversion to C3adesArg and moreover eliminated it by an additional process. The inactivation to C3adesArg seemed to be located predominantly in the proximal periportal region of the liver sinusoid, since C3a was less effective in orthograde perfusions, when C3a first passed the proximal periportal region before reaching the predominant mass of parenchyma as its site of action, than in retrograde perfusions, when it first passed the perivenous area. These data may be evidence for a periportal scavenger mechanism, by which the liver protects itself from systemically released mediators of inflammation that interfere with the local regulation of liver metabolism and hemodynamics.
Cell-Based Reporter Release Assay to Determine the Potency of Proteolytic Bacterial Neurotoxins
(2018)
Despite the implementation of cell-based replacement methods, the mouse lethality assay is still frequently used to determine the activity of botulinum toxin (BoNT) for medical use. One explanation is that due to the use of neoepitope-specific antibodies to detect the cleaved BoNT substrate, the currently devised assays can detect only one specific serotype of the toxin. Recently, we developed a cell-based functional assay, in which BoNT activity is determined by inhibiting the release of a reporter enzyme that is liberated concomitantly with the neurotransmitter from neurosecretory vesicles. In theory, this assay should be suitable to detect the activity of any BoNT serotype. Consistent with this assumption, the current study shows that the stimulus-dependent release of a luciferase from a differentiated human neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-GLuc cells) was inhibited by BoNT-A and-C. Furthermore, this was also inhibited by BoNT-B and tetanus toxin to a lesser extent and at higher concentrations. In order to provide support for the suitability of this technique in practical applications, a dose–response curve obtained with a pharmaceutical preparation of BoNT-A closely mirrored the activity determined in the mouse lethality assay. In summary, the newly established cell-based assay may represent a versatile and specific alternative to the mouse lethality assay and other currently established cell-based assays.
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.
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.
For the five principal prostanoids PGD2, PGE2, PGF2alpha, prostacyclin and thromboxane A2 eight receptors have been identified that belong to the family of G-protein-coupled receptors. They display an overall homology of merely 30%. However, single amino acids in the transmembrane domains such as an Arg in the seventh transmembrane domain are highly conserved. This Arg has been identified as part of the ligand binding pocket. It interacts with the carboxyl group of the prostanoid. The aim of the current study was to analyze the potential role in ligand binding of His-81 in the second transmembrane domain of the rat PGF2alpha receptor, which is conserved among all PGF2alpha receptors from different species. Molecular modeling suggested that this residue is located in close proximity to the ligand binding pocket Arg 291 in the 7th transmembrane domain. The His81 (H) was exchanged by site-directed mutagenesis to Gln (Q), Asp (D), Arg (R), Ala (A) and Gly (G). The receptor molecules were N-terminally extended by a Flag epitope for immunological detection. All mutant proteins were expressed at levels between 50% and 80% of the wild type construct. The H81Q and H81D receptor bound PGF2alpha with 2-fold and 25-fold lower affinity, respectively, than the wild type receptor. Membranes of cells expressing the H81R, H81A or H81G mutants did not bind significant amounts of PGF2alpha. Wild type receptor and H81Q showed a shallow pH optimum for PGF2alpha binding around pH 5.5 with almost no reduction of binding at higher pH. In contrast the H81D mutant bound PGF2alpha with a sharp optimum at pH 4.5, a pH at which the Asp side chain is partially undissociated and may serve as a hydrogen bond donor as do His and Gln at higher pH values. The data indicate that the His-81 in the second transmembrane domain of the PGF2alpha receptor in concert with Arg-291 in the seventh transmembrane domain may be involved in ligand binding, most likely not by ionic interaction with the prostaglandin's carboxyl group but rather as a hydrogen bond donor.
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.
Background and Purpose Recent studies suggested a role for PGE2 in the expression of the chemokine IL-8. PGE2 signals via four different GPCRs, EP1-EP4. The role of EP1 and EP4 receptors for IL-8 induction was studied in HEK293 cells, overexpressing EP1 (HEK-EP1), EP4 (HEK-EP4) or both receptors (HEK-EP1 + EP4). Experimental Approach IL-8 mRNA and protein induction and IL-8 promoter and NF-?B activation were assessed in EP expressing HEK cells. Key Results In HEK-EP1 and HEK-EP1 + EP4 but not HEK or HEK-EP4 cells, PGE2 activated the IL-8 promoter and induced IL-8 mRNA and protein synthesis. Stimulation of HEK-EP1 + EP4 cells with an EP1-specific agonist activated IL-8 promoter and induced IL-8 mRNA and protein, whereas a specific EP4 agonist neither activated the IL-8 promoter nor induced IL-8 mRNA and protein synthesis. Simultaneous stimulation of HEK- EP1 + EP4 cells with both agonists activated IL-8 promoter and induced IL-8 mRNA to the same extent as PGE2. In HEK-EP1 + EP4 cells, PGE2-mediated IL-8 promoter activation and IL-8 mRNA induction were blunted by inhibition of I?B kinase. PGE2 activated NF-?B in HEK-EP1, HEK-EP4 and HEK-EP1 + EP4 cells. In HEK-EP1 + EP4 cells, simultaneous activation of both receptors was needed for maximal PGE2-induced NF-?B activation. PGE2-stimulated NF-?B activation by EP1 was blocked by inhibitors of PLC, calcium-signalling and Src-kinase, whereas that induced by EP4 was only blunted by Src-kinase inhibition. Conclusions and Implications These findings suggest that PGE2-mediated NF-?B activation by simultaneous stimulation of EP1 and EP4 receptors induces maximal IL-8 promoter activation and IL-8 mRNA and protein induction.
Cell-Based Reporter Release Assay to Determine the Potency of Proteolytic Bacterial Neurotoxins
(2018)
Despite the implementation of cell-based replacement methods, the mouse lethality assay is still frequently used to determine the activity of botulinum toxin (BoNT) for medical use. One explanation is that due to the use of neoepitope-specific antibodies to detect the cleaved BoNT substrate, the currently devised assays can detect only one specific serotype of the toxin. Recently, we developed a cell-based functional assay, in which BoNT activity is determined by inhibiting the release of a reporter enzyme that is liberated concomitantly with the neurotransmitter from neurosecretory vesicles. In theory, this assay should be suitable to detect the activity of any BoNT serotype. Consistent with this assumption, the current study shows that the stimulus-dependent release of a luciferase from a differentiated human neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-GLuc cells) was inhibited by BoNT-A and-C. Furthermore, this was also inhibited by BoNT-B and tetanus toxin to a lesser extent and at higher concentrations. In order to provide support for the suitability of this technique in practical applications, a dose–response curve obtained with a pharmaceutical preparation of BoNT-A closely mirrored the activity determined in the mouse lethality assay. In summary, the newly established cell-based assay may represent a versatile and specific alternative to the mouse lethality assay and other currently established cell-based assays.