Using mouse knockouts for mitogen-and stress-activated protein kinase 1 (MSK1) and MSK2 and a double knockout of both MSK1 and MSK2, we show that these protein kinases are required for the stress-induced phosphorylation of transcription factors CREB and ATF1 in primary embryonic fibroblasts. In contrast mitogen-induced phosphorylation of CREB and ATF1 is greatly reduced but not totally abolished. The mitogen-and stress-induced phosphorylation of CREB at Ser133 has been linked to the transcription of several immediate early genes, including c-fos, junB, and egr1. The knockout of both MSK1 and MSK2 resulted in a 50% reduction in c-fos and junB gene transcription in response to anisomycin or UV-C radiation but only a small reduction in response to tetradecanoyl phorbol acetate or epidermal growth factor in fibroblasts. The transcription of egr1 in response to both mitogenic and stress stimuli, as well as stress-induced apoptosis, was unaffected in the MSK1/MSK2 double knockout.Mitogen-activated protein kinase (MAPK) cascades are involved in the transduction of signals from mitogens and cellular stresses into appropriate cellular responses and are required for many functions including cell proliferation, differentiation, and survival (10). One of the ways in which MAPKs produce cellular responses is by the phosphorylation and activation of transcription factors, either directly or indirectly by other protein kinases that they activate. One such transcription factor is the cyclic AMP response element-binding protein, CREB, which requires phosphorylation at Ser133 to become active. Ser133 phosphorylation is induced by stimulating cells with cyclic AMP-elevating agents or mitogens or by exposure to cellular stresses. The cyclic AMP-induced phosphorylation of CREB is catalyzed by cyclic AMP-dependent protein kinase (PKA), but mitogen-induced phosphorylation is prevented by compounds PD-98059, U0126, and PD-184352 (4, 14, 41), which prevent the activation of MAPK kinase 1 (MKK1) and hence block the classical MAPK cascade relatively specifically. At higher concentrations they also inhibit the activation of MKK5 and its substrate extracellular signal-regulated kinase 5 (ERK5) (24,29). In contrast, the stress-induced phosphorylation of CREB is prevented by SB-203580 (14), an inhibitor of another MAPK family member, stress-activated protein kinase 2 (SAPK2), or p38, which is a component of a distinct signal transduction pathway.The phosphorylation of CREB at Ser133 is not catalyzed by MAPK family members directly but by other protein kinases that they activate. Protein kinases that are activated by mitogenic stimuli and that phosphorylate CREB at Ser133 in vitro include the isoforms of MAPK-activated protein kinase 1 (MAPKAP-K1, also called RSK) and mitogen and stress-activated protein kinase (MSK), which are activated by ERK1 and ERK2 of the classical MAPK cascade. However, whether both of these protein kinases or just one of them mediates the phosphorylation of CREB in vivo under different conditions and in different cells...
Metabotropic glutamate receptors are class C G-protein-coupled receptors which respond to the neurotransmitter glutamate. Structural studies have been restricted to the amino-terminal extracellular domain, providing little understanding of the membrane-spanning signal transduction domain. Metabotropic glutamate receptor 5 is of considerable interest as a drug target in the treatment of fragile X syndrome, autism, depression, anxiety, addiction and movement disorders. Here we report the crystal structure of the transmembrane domain of the human receptor in complex with the negative allosteric modulator, mavoglurant. The structure provides detailed insight into the architecture of the transmembrane domain of class C receptors including the precise location of the allosteric binding site within the transmembrane domain and key micro-switches which regulate receptor signalling. This structure also provides a model for all class C G-protein-coupled receptors and may aid in the design of new small-molecule drugs for the treatment of brain disorders.
Protease-activated receptors (PARs) are a family of G-protein-coupled receptors (GPCRs) that are irreversibly activated by proteolytic cleavage of the N terminus, which unmasks a tethered peptide ligand that binds and activates the transmembrane receptor domain, eliciting a cellular cascade in response to inflammatory signals and other stimuli. PARs are implicated in a wide range of diseases, such as cancer and inflammation. PARs have been the subject of major pharmaceutical research efforts but the discovery of small-molecule antagonists that effectively bind them has proved challenging. The only marketed drug targeting a PAR is vorapaxar, a selective antagonist of PAR1 used to prevent thrombosis. The structure of PAR1 in complex with vorapaxar has been reported previously. Despite sequence homology across the PAR isoforms, discovery of PAR2 antagonists has been less successful, although GB88 has been described as a weak antagonist. Here we report crystal structures of PAR2 in complex with two distinct antagonists and a blocking antibody. The antagonist AZ8838 binds in a fully occluded pocket near the extracellular surface. Functional and binding studies reveal that AZ8838 exhibits slow binding kinetics, which is an attractive feature for a PAR2 antagonist competing against a tethered ligand. Antagonist AZ3451 binds to a remote allosteric site outside the helical bundle. We propose that antagonist binding prevents structural rearrangements required for receptor activation and signalling. We also show that a blocking antibody antigen-binding fragment binds to the extracellular surface of PAR2, preventing access of the tethered ligand to the peptide-binding site. These structures provide a basis for the development of selective PAR2 antagonists for a range of therapeutic uses.
Fragment screening of a thermostabilized mGlu5 receptor using a high-concentration radioligand binding assay enabled the identification of moderate affinity, high ligand efficiency (LE) pyrimidine hit 5. Subsequent optimization using structure-based drug discovery methods led to the selection of 25, HTL14242, as an advanced lead compound for further development. Structures of the stabilized mGlu5 receptor complexed with 25 and another molecule in the series, 14, were determined at resolutions of 2.6 and 3.1 Å, respectively.
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