Ligand-Independent G Protein-Coupled Estrogen Receptor/G Protein-Coupled Receptor 30 Activity: Lack of Receptor-Dependent Effects of G-1 and 17ß-Estradiol.

G protein-coupled receptor 30 (GPR30) is a membrane receptor reported to bind 17ß-estradiol (E2) and mediate rapid nongenomic estrogen responses, hence also named G protein-coupled estrogen receptor. G-1 is a proposed GPR30-specific agonist that has been used to implicate the receptor in several pathophysiological events. However, controversy surrounds the role of GPR30 in G-1 and E2 responses. We investigated GPR30 activity in the absence and presence of G-1 and E2 in several eukaryotic systems ex vivo and in vitro in the absence and presence of the receptor. Ex vivo activity was addressed using the caudal artery from wild-type (WT) and GPR30 knockout (KO) mice, and in vitro activity was addressed using a HeLa cell line stably expressing a synthetic multifunctional promoter (nuclear factor κB, signal transducer and activator of transcription, activator protein 1)-luciferase construct (HFF11 cells) and a human GPR30-inducible T-REx system (T-REx HFF11 cells), HFF11 and human embryonic kidney 293 cells transiently expressing WT GPR30 and GPR30 lacking the C-terminal PDZ (postsynaptic density-95/discs-large /zonula occludens-1 homology) motif SSAV, and yeast Saccharomyces cerevisiae transformed to express GPR30. WT and KO arteries exhibited similar contractile responses to 60 mM KCl and 0.3 µM cirazoline, and G-1 relaxed both arteries with the same potency and efficacy. Furthermore, expression of GPR30 did not introduce any responses to 1 µM G-1 and 0.1 µM E2 in vitro. On the other hand, receptor expression caused considerable ligand-independent activity in vitro, which was receptor PDZ motif-dependent in mammalian cells. We conclude from these results that GPR30 exhibits ligand-independent activity in vitro but no G-1- or E2-stimulated activity in any of the systems used. SIGNIFICANCE STATEMENT: Much controversy surrounds 17ß-estradiol (E2) and G-1 as G protein-coupled receptor 30 (GPR30) agonists. We used several recombinant eukaryotic systems ex vivo and in vitro with and without GPR30 expression to address the role of this receptor in responses to these proposed agonists. Our results show that GPR30 exhibits considerable ligand-independent activity in vitro but no G-1- or E2-stimulated activity in any of the systems used. Thus, classifying GPR30 as an estrogen receptor and G-1 as a specific GPR30 agonist is unfounded.

G protein-coupled estrogen receptor 1 (GPER1)/GPR30 increases ERK1/2 activity through PDZ motif-dependent and -independent mechanisms.

G protein-coupled receptor 30 (GPR30), also called G protein-coupled estrogen receptor 1 (GPER1), is thought to play important roles in breast cancer and cardiometabolic regulation, but many questions remain about ligand activation, effector coupling, and subcellular localization. We showed recently that GPR30 interacts through the C-terminal type I PDZ motif with SAP97 and protein kinase A (PKA)-anchoring protein (AKAP) 5, which anchor the receptor in the plasma membrane and mediate an apparently constitutive decrease in cAMP production independently of Gi/o Here, we show that GPR30 also constitutively increases ERK1/2 activity. Removing the receptor PDZ motif or knocking down specifically AKAP5 inhibited the increase, showing that this increase also requires the PDZ interaction. However, the increase was inhibited by pertussis toxin as well as by wortmannin but not by AG1478, indicating that Gi/o and phosphoinositide 3-kinase (PI3K) mediate the increase independently of epidermal growth factor receptor transactivation. FK506 and okadaic acid also inhibited the increase, implying that a protein phosphatase is involved. The proposed GPR30 agonist G-1 also increased ERK1/2 activity, but this increase was only observed at a level of receptor expression below that required for the constitutive increase. Furthermore, deleting the PDZ motif did not inhibit the G-1-stimulated increase. Based on these results, we propose that GPR30 increases ERK1/2 activity via two Gi/o-mediated mechanisms, a PDZ-dependent, apparently constitutive mechanism and a PDZ-independent G-1-stimulated mechanism.

Rrp6 is recruited to transcribed genes and accompanies the spliced mRNA to the nuclear pore.

Rrp6 is an exoribonuclease involved in the quality control of mRNA biogenesis. We have analyzed the association of Rrp6 with the Balbiani ring pre-mRNPs of Chironomus tentans to obtain insight into the role of Rrp6 in splicing surveillance. Rrp6 is recruited to transcribed genes and its distribution along the genes does not correlate with the positions of exons and introns. In the nucleoplasm, Rrp6 is bound to both unspliced and spliced transcripts. Rrp6 is released from the mRNPs in the vicinity of the nuclear pore before nucleo-cytoplasmic translocation. We show that Rrp6 is associated with newly synthesized transcripts during all the nuclear steps of gene expression and is associated with the transcripts independently of their splicing status. These observations suggest that the quality control of pre-mRNA splicing is not based on the selective recruitment of the exoribonuclease Rrp6 to unprocessed mRNAs.

Two types of muscarinic acetylcholine receptors in Drosophila and other arthropods.

Muscarinic acetylcholine receptors (mAChRs) play a central role in the mammalian nervous system. These receptors are G protein-coupled receptors (GPCRs), which are activated by the agonists acetylcholine and muscarine, and blocked by a variety of antagonists. Mammals have five mAChRs (m1-m5). In this study, we cloned two structurally related GPCRs from the fruit fly Drosophila melanogaster, which, after expression in Chinese hamster ovary cells, proved to be muscarinic acetylcholine receptors. One mAChR (the A-type; encoded by gene CG4356) is activated by acetylcholine (EC50, 5 × 10(-8) M) and muscarine (EC50, 6 × 10(-8) M) and blocked by the classical mAChR antagonists atropine, scopolamine, and 3-quinuclidinyl-benzilate (QNB), while the other (the B-type; encoded by gene CG7918) is also activated by acetylcholine, but has a 1,000-fold lower sensitivity to muscarine, and is not blocked by the antagonists. A- and B-type mAChRs were also cloned and functionally characterized from the red flour beetle Tribolium castaneum. Recently, Haga et al. (Nature 2012, 482: 547-551) published the crystal structure of the human m2 mAChR, revealing 14 amino acid residues forming the binding pocket for QNB. These residues are identical between the human m2 and the D. melanogaster and T. castaneum A-type mAChRs, while many of them are different between the human m2 and the B-type receptors. Using bioinformatics, one orthologue of the A-type and one of the B-type mAChRs could also be found in all other arthropods with a sequenced genome. Protostomes, such as arthropods, and deuterostomes, such as mammals and other vertebrates, belong to two evolutionarily distinct lineages of animal evolution that split about 700 million years ago. We found that animals that originated before this split, such as cnidarians (Hydra), had two A-type mAChRs. From these data we propose a model for the evolution of mAChRs.

Identification of the Drosophila and Tribolium receptors for the recently discovered insect RYamide neuropeptides.

One year ago, we discovered a new family of insect RYamide neuropeptides, which has the C-terminal consensus sequence FFXXXRYamide, and which is widely occurring in most insects, including the fruitfly Drosophila melanogaster and the red flour beetle Tribolium castaneum (F. Hauser et al., J. Proteome Res. 9 (2010) 5296-5310). Here, we identify a Drosophila G-protein-coupled receptor (GPCR) coded for by gene CG5811 and its Tribolium GPCR ortholog as insect RYamide receptors. The Drosophila RYamide receptor is equally well activated (EC(50), 1×10(-9)M) by the two Drosophila RYamide neuropeptides: RYamide-1 (PVFFVASRYamide) and RYamide-2 (NEHFFLGSRYamide), both contained in a preprohormone coded for by gene CG40733. The Tribolium receptor shows a somewhat higher affinity to Tribolium RYamide-2 (ADAFFLGPRYamide; EC(50), 5×10(-9)M) than to Tribolium RYamide-1 (VQNLATFKTMMRYamide; EC(50), 7×10(-8)M), which might be due to the fact that the last peptide does not completely follow the RYamide consensus sequence rule. There are other neuropeptides in insects that have similar C-terminal sequences (RWamide or RFamide), such as the FMRFamides, sulfakinins, myosuppressins, neuropeptides F, and the various short neuropeptides F. Amazingly, these neuropeptides show no cross-reactivity to the Tribolium RYamide receptor, while the Drosophila RYamide receptor is only very slightly activated by high concentrations (>10(-6)M) of neuropeptide F and short neuropeptide F-1, showing that the two RYamide receptors are quite specific for activation by insect RYamides, and that the sequence FFXXXRYamide is needed for effective insect RYamide receptor activation. Phylogenetic tree analyses and other amino acid sequence comparisons show that the insect RYamide receptors are not closely related to any other known insect or invertebrate/vertebrate receptors, including mammalian neuropeptide Y and insect neuropeptide F and short neuropeptide F receptors. Gene expression data published in Flybase (www.flybase.org) show that the Drosophila CG5811 gene is significantly expressed in the hindgut of adult flies, suggesting a role of insect RYamides in digestion or water reabsorption.

Surface display of Salmonella epitopes in Escherichia coli and Staphylococcus carnosus.

BACKGROUND: Salmonella enterica serotype Enteritidis (SE) is considered to be one of the most potent pathogenic Salmonella serotypes causing food-borne disease in humans. Since a live bacterial vaccine based on surface display of antigens has many advantages over traditional vaccines, we have studied the surface display of the SE antigenic proteins, H:gm and SefA in Escherichia coli by the ß-autotransporter system, AIDA. This procedure was compared to protein translocation in Staphylococcus carnosus, using a staphylococci hybrid vector earlier developed for surface display of other vaccine epitopes. RESULTS: Both SefA and H:gm were translocated to the outer membrane in Escherichia coli. SefA was expressed to full length but H:gm was shorter than expected, probably due to a proteolytic cleavage of the N-terminal during passage either through the periplasm or over the membrane. FACS analysis confirmed that SefA was facing the extracellular environment, but this could not be conclusively established for H:gm since the N-terminal detection tag (His6) was cleaved off. Polyclonal salmonella antibodies confirmed the sustained antibody-antigen binding towards both proteins. The surface expression data from Staphylococcus carnosus suggested that the H:gm and SefA proteins were transported to the cell wall since the detection marker was displayed by FACS analysis. CONCLUSION: Apart from the accumulated knowledge and the existence of a wealth of equipment and techniques, the results indicate the selection of E. coli for further studies for surface expression of salmonella antigens. Surface expression of the full length protein facing the cell environment was positively proven by standard analysis, and the FACS signal comparison to expression in Staphylococcus carnosus shows that the distribution of the surface protein on each cell was comparatively very narrow in E. coli, the E. coli outer membrane molecules can serve as an adjuvant for the surface antigenic proteins and multimeric forms of the SefA protein were detected which would probably be positive for the realisation of a strong antigenic property. The detection of specific and similar proteolytic cleavage patterns for both the proteins provides a further starting point for the investigation and development of the Escherichia coli AIDA autotransporter efficiency.

Human G protein-coupled receptor 30 is N-glycosylated and N-terminal domain asparagine 44 is required for receptor structure and activity.

G protein-coupled receptor 30 (GPR30), or G protein-coupled estrogen receptor (GPER), is a G protein-coupled receptor (GPCR) that is currently attracting considerable attention in breast cancer and cardiometabolic regulation. The receptor was reported to be a novel membrane estrogen receptor mediating rapid non-genomic responses. However, questions remain about both the cognate ligand and the subcellular localization of receptor activity. Here, we used human embryonic kidney (HEK) 293 (HEK293) cells ectopically expressing N-terminally FLAG-tagged human GPR30 and three unique antibodies (Ab) specifically targetting the receptor N-terminal domain (N-domain) to investigate the role of N-glycosylation in receptor maturation and activity, the latter assayed by constitutive receptor-stimulated extracellular-regulated protein kinase (ERK) 1/2 (ERK1/2) activity. GPR30 expression was complex with receptor species spanning from approximately 40 kDa to higher molecular masses and localized in the endoplasmatic reticulum (ER), the plasma membrane (PM), and endocytic vesicles. The receptor contains three conserved asparagines, Asn25, Asn32, and Asn44, in consensus N-glycosylation motifs, all in the N-domain, and PNGase F treatment showed that at least one of them is N-glycosylated. Mutating Asn44 to isoleucine inactivated the receptor, yielding a unique receptor species at approximately 20 kDa that was recognized by Ab only in a denatured state. On the other hand, mutating Asn25 or Asn32 either individually or in combination, or truncating successively N-domain residues 1-42, had no significant effect either on receptor structure, maturation, or activity. Thus, Asn44 in the GPR30 N-domain is required for receptor structure and activity, whereas N-domain residues 1-42, including specifically Asn25 and Asn32, do not play any major structural or functional role(s).

Transient erythromycin resistance phenotype associated with peptidyl-tRNA drop-off on early UGG and GGG codons.

Expression of minigenes encoding tetra- or pentapeptides MXLX or MXLXV (E peptides), where X is a nonpolar amino acid, renders cells erythromycin resistant whereas expression of minigenes encoding tripeptide MXL does not. By using a 3A' reporter gene system beginning with an E-peptide-encoding sequence, we asked whether the codons UGG and GGG, which are known to promote peptidyl-tRNA drop-off at early positions in mRNA, would result in a phenotype of erythromycin resistance if located after this sequence. We find that UGG or GGG, at either position +4 or +5, without a following stop codon, is associated with an erythromycin resistance phenotype upon gene induction. Our results suggest that, while a stop codon at +4 gives a tripeptide product (MIL) and erythromycin sensitivity, UGG or GGG codons at the same position give a tetrapeptide product (MILW or MILG) and phenotype of erythromycin resistance. Thus, the drop-off event on GGG or UGG codons occurs after incorporation of the corresponding amino acid into the growing peptide chain. Drop-off gives rise to a peptidyl-tRNA where the peptide moiety functionally mimics a minigene peptide product of the type previously associated with erythromycin resistance. Several genes in Escherichia coli fulfill the requirements of high mRNA expression and an E-peptide sequence followed by UGG or GGG at position +4 or +5 and should potentially be able to give an erythromycin resistance phenotype.

A codon window in mRNA downstream of the initiation codon where NGG codons give strongly reduced gene expression in Escherichia coli.

The influences on gene expression by codons at positions +2, +3, +5 and +7 downstream of the initiation codon have been compared. Most of the +2 codons that are known to give low gene expression are associated with a higher expression if placed at the later positions. The NGG codons AGG, CGG, UGG and GGG, but not GGN or GNG (where N is non-G), are unique since they are associated with a very low gene expression also if located at positions +2, +3 and +5. All codons, including NGG, give a normal gene expression if placed at positions +7. The negative effect by the NGG codons is true for both the lacZ and 3A' model genes. The low expression is suggested to originate at the translational level, although it is not the result of mRNA secondary structure or a lowered intracellular mRNA pool.

Abortive translation caused by peptidyl-tRNA drop-off at NGG codons in the early coding region of mRNA.

In Escherichia coli the codons CGG, AGG, UGG or GGG (NGG codons) but not GGN or GNG (where N is non-G) are associated with low expression of a reporter gene, if located at positions +2 to +5. Induction of a lacZ reporter gene with any one of the NGG codons at position +2 to +5 does not influence growth of a normal strain, but growth of a strain with a defective peptidyl-tRNA hydrolase (Pth) enzyme is inhibited. The same codons, if placed at position +7, did not give this effect. Other codons, such as CGU and AGA, at location +2 to +5, did not give any growth inhibition of either the wild-type or the mutant strain. The inhibitory effect on the pth mutant strain by NGG codons at location +5 was suppressed by overexpression of the Pth enzyme from a plasmid. However, the overexpression of cognate tRNAs for AGG or GGG did not rescue from the growth inhibition associated with these codons early in the induced model gene. The data suggest that the NGG codons trigger peptidyl-tRNA drop-off if located at early coding positions in mRNA, thereby strongly reducing gene expression. This does not happen if these codons are located further down in the mRNA at position +7, or later.

The incorporation of 5-fluorouracil into RNA affects the ribonucleolytic activity of the exosome subunit Rrp6.

5-Fluorouracil (5FU) is a fluoropyrimidine used for the treatment of solid tumors. 5FU is a precursor of dTTP and UTP during biogenesis, and it interferes with both DNA and RNA metabolism. The RNA exosome, a multisubunit complex with ribonucleolytic activity, has been identified as one of the targets of 5FU in yeast. Studies in human cells have shown that the catalytic subunit of the nuclear exosome, Rrp6, is specifically targeted. Here, we have investigated the direct effect of 5FU on the activity of Rrp6 in Drosophila S2 cells, and we have identified two aspects of Rrp6 function that are altered by 5FU. First, gel filtration analysis revealed that the repertoire of multimolecular complexes that contain Rrp6 is modified by exposure to 5FU, which is consistent with the proposal that incorporation of 5FU into RNA leads to the sequestration of Rrp6 in ribonucleoprotein complexes. Second, the incorporation of 5FU into RNA renders the RNA less susceptible to degradation by Rrp6, as shown by Rrp6 activity assays in vitro. Our results imply that aberrant transcripts synthesized in 5FU-treated cells cannot be turned over efficiently by the surveillance machinery. Together with previous results on the mechanisms of action of 5FU, our findings suggest that the cytotoxicity of 5FU at the RNA level is the result of at least three different effects: the increased levels of retroviral transcripts with mutagenic potential, the reduced synthesis of ribosomes, and the inhibition of the nuclear RNA surveillance pathways. Drugs that reinforce any of these effects may boost the cytotoxicity of 5FU.

The exosome associates cotranscriptionally with the nascent pre-mRNP through interactions with heterogeneous nuclear ribonucleoproteins.

Eukaryotic cells have evolved quality control mechanisms to degrade aberrant mRNA molecules and prevent the synthesis of defective proteins that could be deleterious for the cell. The exosome, a protein complex with ribonuclease activity, is a key player in quality control. An early quality checkpoint takes place cotranscriptionally but little is known about the molecular mechanisms by which the exosome is recruited to the transcribed genes. Here we study the core exosome subunit Rrp4 in two insect model systems, Chironomus and Drosophila. We show that a significant fraction of Rrp4 is associated with the nascent pre-mRNPs and that a specific mRNA-binding protein, Hrp59/hnRNP M, interacts in vivo with multiple exosome subunits. Depletion of Hrp59 by RNA interference reduces the levels of Rrp4 at transcription sites, which suggests that Hrp59 is needed for the exosome to stably interact with nascent pre-mRNPs. Our results lead to a revised mechanistic model for cotranscriptional quality control in which the exosome is constantly recruited to newly synthesized RNAs through direct interactions with specific hnRNP proteins.

Influences on gene expression in vivo by a Shine-Dalgarno sequence.

The Shine-Dalgarno (SD+: 5'-AAGGAGG-3') sequence anchors the mRNA by base pairing to the 16S rRNA in the small ribosomal subunit during translation initiation. We have here compared how an SD+ sequence influences gene expression, if located upstream or downstream of an initiation codon. The positive effect of an upstream SD+ is confirmed. A downstream SD+ gives decreased gene expression. This effect is also valid for appropriately modified natural Escherichia coli genes. If an SD+ is placed between two potential initiation codons, initiation takes place predominantly at the second start site. The first start site is activated if the distance between this site and the downstream SD+ is enlarged and/or if the second start site is weakened. Upstream initiation is eliminated if a stable stem-loop structure is placed between this SD+ and the upstream start site. The results suggest that the two start sites compete for ribosomes that bind to an SD+ located between them. A minor positive contribution to upstream initiation resulting from 3' to 5' ribosomal diffusion along the mRNA is suggested. Analysis of the E. coli K12 genome suggests that the SD+ or SD-like sequences are systematically avoided in the early coding region suggesting an evolutionary significance.