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Negative allosteric modulation of the metabotropic glutamate 5 (mGlu5) receptor has emerged as a potential strategy for the treatment of neurological disorders. Despite the success in preclinical studies, many mGlu5 negative allosteric modulators (NAMs) that have reached clinical trials failed due to lack of efficacy. In this study, we provide a detailed in vitro pharmacological characterization of nine clinically and preclinically tested NAMs. We evaluated inhibition of L-glutamate-induced signaling with Ca2+ mobilization, inositol monophosphate (IP1) accumulation, extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, and real-time receptor internalization assays on rat mGlu5 expressed in HEK293A cells. Moreover, we determined association (kon) and dissociation (koff) rates, as well as NAM affinity with [3H]methoxy-PEPy binding experiments. kon and koff values varied greatly between the nine NAMs (34- and 139-fold, respectively) resulting in long receptor residence times (>400 min) for basimglurant and mavoglurant, medium residence times (10-30 min) for AZD2066, remeglurant and (RS)-remeglurant, and low residence time (<10 min) for dipraglurant, F169521, F1699611 and STX107. We found that all NAMs inhibited L-glutamate induced mGlu5 receptor internalization, generally with a similar potency to IP1 accumulation and ERK1/2 phosphorylation while Ca2+ mobilization was less potently inhibited. Operational model of allosterism analyses revealed that dipraglurant and (RS)-remeglurant were biased towards (affinity) receptor internalization and away (cooperativity) from the ERK1/2 phosphorylation pathway, respectively. Our study is the first to measure binding kinetics and negative allosteric modulation of mGlu5 receptor internalization, and adds significant new knowledge about the molecular pharmacology of a diverse range of clinically relevant NAMs. SIGNIFICANCE STATEMENT: The metabotropic glutamate 5 (mGlu5) receptor is important in many brain func

BACKGROUND: We hypothesized that single nucleotide polymorphisms (SNPs) located in microRNA (miR) binding sites in genes of the renin angiotensin aldosterone system (RAAS) can influence blood pressure and risk of myocardial infarction. METHODS: Using online databases dbSNP and TargetScan, we identified 10 SNPs in potential miR binding sites in eight RAAS-related genes, common in Caucasians. We genotyped a large case-control study on myocardial infarctions, the Study of Myocardial Infarctions LEiden (SMILE) for these 10 SNPs and found nine SNPs, in seven genes, to be prevalent. Functionality of each SNP in interfering with mRNA/miR binding was tested using a dual luciferase reporter gene system. RESULTS: Of these nine SNPs, four SNPs, located in the arginine vasopressin 1A receptor (AVPR1A), bradykinin 2 receptor (BDKRB2), and thromboxane A2 receptor (TBXA2R) genes were associated with blood pressure. The rare allele of the AVPR1A SNP rs11174811, was associated with increased blood pressure whereas the rare alleles of the two linked BDKRB2 SNPs rs5225 and rs2069591 and of the TBXA2R SNP rs13306046 were associated with decreased blood pressure. Although not associated with blood pressure, the rare allele of the mineralocorticoid receptor (NR3C2) SNP rs5534, was associated with a twofold increased risk of myocardial infarction in men younger than 50 years. For all of these five SNPs, except rs2069591, we could demonstrate a reduction in miR-induced repression of gene expression. CONCLUSIONS: Common SNPs in miR binding sites of RAAS-related genes can influence both blood pressure and risk of myocardial infarction. These results may imply an important role for SNPs in miR target sites in human disease.

Seven-transmembrane receptors (7TMRs) signal through the well described heterotrimeric G proteins but can also activate G protein-independent signaling pathways of which the impact and complexity are less understood. The angiotensin II type 1 receptor (AT(1)R) is a prototypical 7TMR and an important drug target in cardiovascular diseases. "Biased agonists" with intrinsic "functional selectivity" that simultaneously blocks Galpha(q) protein activity and activates G protein-independent pathways of the AT(1)R confer important perspectives in treatment of cardiovascular diseases. In this study, we performed a global quantitative phosphoproteomics analysis of the AT(1)R signaling network. We analyzed ligand-stimulated SILAC (stable isotope labeling by amino acids in cell culture) cells by high resolution (LTQ-Orbitrap) MS and compared the phosphoproteomes of the AT(1)R agonist angiotensin II and the biased agonist [Sar(1),Ile(4),Ile(8)]angiotensin II (SII angiotensin II), which only activates the Galpha(q) protein-independent signaling. We quantified more than 10,000 phosphorylation sites of which 1183 were regulated by angiotensin II or its analogue SII angiotensin II. 36% of the AT(1)R-regulated phosphorylations were regulated by SII angiotensin II. Analysis of phosphorylation site patterns showed a striking distinction between protein kinases activated by Galpha(q) protein-dependent and -independent mechanisms, and we now place protein kinase D as a key protein involved in both Galpha(q)-dependent and -independent AT(1)R signaling. This study provides substantial novel insight into angiotensin II signal transduction and is the first study dissecting the differences between a full agonist and a biased agonist from a 7TMR on a systems-wide scale. Importantly, it reveals a previously unappreciated diversity and quantity of Galpha(q) protein-independent signaling and uncovers novel signaling pathways. We foresee that the amount and diversity of G protein-independent signa

Seven-transmembrane receptors (7TMRs) signal through the well described heterotrimeric G proteins, but can also activate G protein-independent signaling pathways of which the impact and complexity are less understood. The Angiotensin II type 1 receptor (AT1R) is a prototypical 7TMR and an important drug target in cardiovascular diseases. "Biased agonists", with intrinsic "functional selectivity", that simultaneously blocks Gaq protein activity, and activitates G protein independent pathways of the AT1R, confer important perspectives in treatment of cardiovascular diseases.n this study we performed a global quantitative phosphoproteomics analysis of the AT1R signaling network. We analyzed ligand-stimulated SILAC cells by high-resolution mass spectrometry (LTQ Orbitrap MS) and compared the phosphoproteomes of the AT1R agonist Angiotensin II and the biased agonist SII Angiotensin II, which only activates the Gaq protein-independent signaling.e quantified more than ten thousand phosphorylation sites of which 1183 were regulated by Angiotensin II or its analogue SII Angiotensin II. 36% of the AT1R regulated phosphorylations were regulated by SII Angiotensin II. Analysis of phosphorylation site patterns displays a striking distinction between protein kinases activated by Gaq protein-dependent and -independent mechanisms, and we now place protein kinase D as a key protein involved in both Gaq-dependent and independent AT1R signaling.his study provides substantial novel insight into Angiotensin II signal transduction and is the first study dissecting the differences between a full agonist and a biased agonist from a 7TMR on a systems-wide scale. Importantly, it reveals a previously unappreciated diversity and quantity of Gaq protein-independent signaling and uncovers novel signaling pathways. We foresee that the amount and diversity of G protein independent signaling may be more pronounced than previously recognized for other 7TMRs as well. Quantitative mass spectrometry is

Udgivelsesdato: 2009-May, Residues that mediate helix-helix interactions within the seven transmembranes (TM) of G protein-coupled receptors are important for receptor biogenesis and the receptor switch mechanism. By contrast, the residues directly contacting the lipid bilayer have only recently garnered attention as potential receptor dimerization interfaces. In the present study, we aimed to determine the contributions of these lipid-facing residues to receptor function and oligomerization by systemically generating chimeric complement factor 5a receptors in which the entire lipid-exposed surface of a single TM helix was exchanged with the cognate residues from the angiotensin type 1 receptor. Disulfide-trapping and bioluminescence resonance energy transfer (BRET) studies demonstrated robust homodimerization of both complement factor 5a receptor and angiotensin type 1 receptor, but no evidence for heterodimerization. Despite relatively conservative substitutions, the lipid-facing chimeras (TM1, TM2, TM4, TM5, TM6 or TM7) were retained in the endoplasmic reticulum/cis-Golgi network. With the exception of the TM7 chimera that did not bind ligand, the lipid-facing chimeras bound ligand with low affinity, but similar to wild-type complement factor 5a receptors trapped in the endoplasmic reticulum with brefeldin A. These results suggest that the chimeric receptors were properly folded; moreover, native complement factor 5a receptors are not fully competent to bind ligand when present in the endoplasmic reticulum. BRET oligomerization studies demonstrated energy transfer between the wild-type complement factor 5a receptor and the lipid-facing chimeras, suggesting that the lipid-facing residues within a single TM segment are not essential for oligomerization. These studies highlight the importance of the lipid-facing residues in the complement factor 5a receptor for transport competence.

Udgivelsesdato: 2009-Jan-16, It has been suggested previously ( AbdAlla, S., Lother, H., and Quitterer, U. (2000) Nature 407, 94-98 ) that the angiotensin II type 1 receptor (AT1R) and the bradykinin B2 receptor (B2R) form constitutive heterodimers. Furthermore they demonstrate that AT1R signaling significantly increases in the presence of the B2R. These findings suggest that heterodimerization and potentiation of AT1R signaling is a universal phenomenon that occurs as a natural consequence of simultaneous expression of the two receptors. Hence this potential interaction is of great pharmacological and biological interest that adds an additional layer of complexity to the understanding of the cross-talk between the renin-angiotensin and kallikrein-kinin systems. Given the remarkable significance of this finding, scientists from four independent research groups have set out to reproduce and further examine the potential AT1R/B2R interaction. We have investigated functional potentiation by the B2R of AT1R signaling in three different cell lines using multiple assays including phosphoinositide hydrolysis, ERK activation, beta-arrestin recruitment, and receptor selection and amplification technology, and we have examined dimerization using bioluminescence resonance energy transfer and regulated secretion/aggregation technology. However, although both the AT1Rs and B2Rs were functional in our systems and the systems were fine tuned to detect small changes in receptor function, we failed to detect any functional modulation by or physical interaction between the two receptor proteins. In contrast to the previous observations, our data collectively suggest that AT1R/B2R heterodimerization does not occur as a natural consequence of their simultaneous expression in the same cell nor does the B2R influence the AT1R signaling.

Udgivelsesdato: 2009-Apr-29, The Renin-Angiotensin System (RAS) is important for the regulation of cardiovascular physiology, where it controls blood pressure, and salt- and water homeostasis. Dysregulation of RAS can lead to severe diseases including hypertension, diabetic nephropathy, and cardiac arrhythmia, and -failure. The importance of the RAS is clearly emphasised by the widespread use of drugs targeting this system in clinical practice. These include, renin inhibitors, angiotensin II receptor type I blockers, and inhibitors of the angiotensin converting enzyme. Some of the important effectors within the system are 7 transmembrane (7TM) receptors (or G-protein-coupled receptors) such as the angiotensin II Receptors type I and II (AT1R and AT2R) and the MAS-oncogene receptor. Several findings indicate that the 7TM receptors can form both homo- and heterodimers, or higher orders of oligomers. Furthermore, dimerization may be important for receptor function, and in the development of cardiovascular diseases. This is very significant, since "dimers" may provide pharmacologists with novel targets for improved drug therapy. However, we know that 7TM receptors can mediate signals as monomeric units, and so far it has been very difficult to establish if our observations reflect actual well-defined dimerization or merely reflect close proximity between the receptors and/or various types of functional interaction. In this review, we will present and critically discuss the current data on 7TM receptor dimerization with a clear focus on the RAS, and delineate future challenges within the field.

Udgivelsesdato: 2008-Feb, To analyze the interaction between the neurokinin-1 (NK-1) receptor and G-protein coupled receptor kinases (GRKs), we performed bioluminescence resonance energy transfer(2) (BRET(2)) measurements between the family A NK-1 receptor and GRK2 and GRK5 as well as their respective kinase-inactive mutants. We observed agonist induced interaction of both GRK5 and GRK2 with the activated NK-1 receptor. In saturation experiments, we observed GRK5 to interact with the activated receptor in a monophasic manner while GRK2 interacted in a biphasic manner with the low affinity phase corresponding to receptor affinity for GRK5. Agonist induced GRK5 interaction with the receptor was dependent on intact kinase-activity, whereas the high affinity phase of GRK2 interaction was independent of kinase activity. We were surprised to find that the BRET(2) saturation experiments indicated that before receptor activation, the full-length NK-1 receptor, but not a functional C-terminal tail-truncated receptor, is preassociated with GRK5 in a relatively low-affinity state. We demonstrate that GRK5 can compete for agonist induced GRK2 interaction with the NK-1 receptor, whereas GRK2 does not compete for receptor interaction with GRK5. We suggest that GRK5 is preassociated with the NK-1 receptor and that GRK5, rather than GRK2, is a key player in competitive regulation of GRK subtype specific interaction with the NK-1 receptor.