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.

Salmonella enterica, the cause of food poisoning and typhoid fever, has evolved sophisticated mechanisms to modulate Rho family guanosine triphosphatases (GTPases) to mediate specific cellular responses such as actin remodeling, macropinocytosis, and nuclear responses. These responses are largely the result of the activity of a set of bacterial proteins (SopE, SopE2, and SopB) that, upon delivery into host cells via a type III secretion system, activate specific Rho family GTPases either directly (SopE and SopE2) or indirectly (SopB) through the stimulation of an endogenous exchange factor. We show that different Rho family GTPases play a distinct role in Salmonella-induced cellular responses. In addition, we report that SopB stimulates cellular responses by activating SH3-containing guanine nucleotide exchange factor (SGEF), an exchange factor for RhoG, which we found plays a central role in the actin cytoskeleton remodeling stimulated by Salmonella. These results reveal a remarkable level of complexity in the manipulation of Rho family GTPases by a bacterial pathogen.

SUMMARY Many bacterial pathogens and symbionts utilize type III secretion systems to deliver bacterial effector proteins into host cells. These effector proteins have the capacity to modulate a large variety of cellular functions in a highly regulated manner. Here we report that the phosphoinositide phosphatase SopB, a Salmonella Typhimurium type III secreted effector protein, diversifies its function by localizing to different cellular compartments in a ubiquitin-dependent manner. We show that SopB utilizes the same enzymatic activity to modulate actin-mediated bacterial internalization and Akt activation at the plasma membrane, and vesicular traffic and intracellular bacterial replication at the phagosome. Thus by exploiting the host cellular machinery, Salmonella Typhimurium has evolved the capacity to broaden the functional repertoire of a virulence factor to maximize its ability to modulate cellular functions.

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A prospective randomized controlled trial was performed to compare the effects of ibuprofen with indomethacin on cerebral hemodynamics measured using near infrared spectroscopy in preterm infants during treatment for patent ductus arteriosus. Infants were randomly assigned to three intravenous doses of either indomethacin (0.20-0.25 mg/kg, 12 hourly) or ibuprofen (5-10 mg/kg, 24 hourly) and also received a dose of saline. The primary end points of the study were the effects of the first dose on cerebral blood flow (CBF) and cerebral blood volume. Fifteen infants received indomethacin and 18 received ibuprofen. The group mean (SD) values for CBF (mL x 100 g(-1) x min(-1)) before and after the first dose of indomethacin were 13.6 (4.1) and 8.3 (3.1), respectively, the change being significant (p<0.001). In contrast, no significant changes in CBF were observed with the first dose of ibuprofen, the respective before and after values being 13.3 (3.2) and 14.9 (4.7) mL x 100 g(-1) x min(-1). The median (interquartile range) value for change in cerebral blood volume (mL/100 g) after the first dose in the indomethacin group was -0.4 (-0.3 to -0.6) and in the ibuprofen group was 0.0 (0.1 to -0.1), the difference between the two groups being significant (p<0.001). Cerebral oxygen delivery changed significantly after the first dose in the indomethacin group but not in the ibuprofen group. Significant reductions in CBF, cerebral blood volume, and cerebral oxygen delivery also occurred after the 24-h dose of indomethacin, but there were no significant changes after the 48-h dose of saline in the indomethacin group or after the 24- and 48-h doses of ibuprofen. The patent ductus arteriosus closure rates after indomethacin and ibuprofen were 93 and 78%, respectively. We conclude that ibuprofen, unlike indomethacin, has no adverse effects on cerebral hemodynamics and appears to mediate patent ductus arteriosus closure.

Phagocytosis is the process whereby cells direct the spatially localized, receptor-driven engulfment of particulate materials. It proceeds via remodeling of the actin cytoskeleton and shares many of the core cytoskeletal components involved in adhesion and migration. Small GTPases of the Rho family have been widely implicated in coordinating actin dynamics in response to extracellular signals and during diverse cellular processes, including phagocytosis, yet the mechanisms controlling their recruitment and activation are not known. We show herein that in response to ligation of Fc receptors for IgG (Fc␥R), the guanine nucleotide exchange factor Vav translocates to nascent phagosomes and catalyzes GTP loading on Rac, but not Cdc42. The Vav-induced Rac activation proceeds independently of Cdc42 function, suggesting distinct roles for each GTPase during engulfment. Moreover, inhibition of Vav exchange activity or of Cdc42 activity does not prevent Rac recruitment to sites of particle attachment. We conclude that Rac is recruited to Fc␥ membrane receptors in its inactive, GDP-bound state and that Vav regulates phagocytosis through subsequent catalysis of GDP/GTP exchange on Rac.

αMβ2 integrins mediate phagocytosis of opsonized particles in a process controlled by RhoA, Rho kinase, myosin II, Arp2/3, and actin polymerization. αMβ2, Rho, Arp2/3, and F-actin accumulate underneath bound particles; however, the mechanism regulating Rho function during αMβ2-mediated phagocytosis is poorly understood. We report that the binding of C3bi-opsonized sheep red blood cells (RBCs) to αMβ2 increases Rho-GTP, but not Rac-GTP, levels. Deletion of the cytoplasmic domain of β2, but not of αM, abolished Rho recruitment and activation, as well as phagocytic uptake. Interestingly, a 16–amino acid (aa) region in the membrane-proximal half of the β2 cytoplasmic domain was necessary for activating Rho. Three COOH-terminal residues (aa 758–760) were essential for β2-induced accumulation of Rho at complement receptor 3 (CR3) phagosomes. Activation of Rho was necessary, but not sufficient, for its stable recruitment underneath bound particles or for uptake. However, recruitment of active Rho was sufficient for phagocytosis. Our data shed light on the mechanism of outside-in signaling, from ligated integrins to the activation of Rho GTPase signaling.

Peptide agonists of the glucagon-like peptide-1 receptor (GLP-1R) have revolutionized diabetes therapy, but their use has been limited because they require injection. Herein, we describe the discovery of the orally bioavailable, small-molecule, GLP-1R agonist PF-06882961 (danuglipron). A sensitized high-throughput screen was used to identify 5-fluoropyrimidine-based GLP-1R agonists that were optimized to promote endogenous GLP-1R signaling with nanomolar potency. Incorporation of a carboxylic acid moiety provided considerable GLP-1R potency gains with improved off-target pharmacology and reduced metabolic clearance, ultimately resulting in the identification of danuglipron. Danuglipron increased insulin levels in primates but not rodents, which was explained by receptor mutagensis studies and a cryogenic electron microscope structure that revealed a binding pocket requiring a primate-specific tryptophan 33 residue. Oral administration of danuglipron to healthy humans produced dose-proportional increases in systemic exposure (NCT03309241). This opens an opportunity for oral small-molecule therapies that target the well-validated GLP-1R for metabolic health.