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Mammalian target of rapamycin complex 2 (mTORC2) phosphorylates AGC protein kinases including protein kinase C (PKC) and regulates cellular functions such as cell migration. However, its regulation remains poorly understood. Here we show that lysophosphatidic acid (LPA) induces two phases of PKC-δ hydrophobic motif phosphorylation. The late phase is mediated by Gα(12), which specifically activates ARAF, leading to upregulation of the RFFL E3 ubiquitin ligase and subsequent ubiquitylation and degradation of the PRR5L subunit of mTORC2. Destabilization of PRR5L, a suppressor of mTORC2-mediated hydrophobic motif phosphorylation of PKC-δ, but not AKT, results in PKC-δ hydrophobic motif phosphorylation and activation. This Gα(12)-mediated signalling pathway for mTORC2 regulation is critically important for fibroblast migration and pulmonary fibrosis development.

Regulator of G-protein signaling 9-2 (RGS9-2), a member of the RGS family of Gα GTPase accelerating proteins, is expressed specifically in the striatum, which participates in antipsychotic-induced tardive dyskinesia and in levodopa-induced dyskinesia. We report that RGS9 knock-out mice develop abnormal involuntary movements when inhibition of dopaminergic transmission is followed by activation of D2-like dopamine receptors (DRs). These abnormal movements resemble drug-induced dyskinesia more closely than other rodent models. Recordings from striatal neurons of these mice establish that activation of D2-like DRs abnormally inhibits glutamate-elicited currents. We show that RGS9-2, via its DEP domain (for Disheveled, EGL-10, Pleckstrin homology), colocalizes with D2DRs when coexpressed in mammalian cells. Recordings from oocytes coexpressing D2DR or the m2 muscarinic receptor and G-protein-gated inward rectifier potassium channels show that RGS9-2, via its DEP domain, preferentially accelerates the termination of D2DR signals. Thus, alterations in RGS9-2 may be a key factor in the pathway leading from D2DRs to the side effects associated with the treatment both of psychoses and Parkinson's disease. [ABSTRACT FROM AUTHOR]

Examines the integration of G[sub 12] and G[sub 13] heterotrimeric G-proteins signaling pathways. Role of G[sub 13] on blood vessel development; Mediation of cell migratory response to lysophosphatidic acid in embryonic fibroblast cells; Use of immunohistochemistry analysis.

The mammalian G proteins G15 and G16 couple a wide variety of receptors to phospholipase C (PLC) in co-transfected systems, and it has been suggested that they can be used as tools in agonist-screening systems. Using the reversed tetracycline-controlled transactivation system we generated rat pituitary GH3 cell clones that expressed Gα15 and Gα16 conditionally to study the coupling of endogenous receptors to both G proteins. In cells expressing moderate levels of Gα15, activation of various endogenous receptors increased inositol phosphate production, whereas conditional expression of Gα16 had no significant effect on agonist-dependent PLC activity. Activation of PLC through Gα15 in response to carbachol did not increase cytosolic [Ca2+] ([Ca2+]i) but stimulated protein kinase C. While carbachol decreased the secretory activity in non-induced GH3 cells, it increased secretion in cells expressing Gα15. Our data demonstrate that Gα15 has a higher functional promiscuity than Gα16 when studied in a system that preserves physiological G protein and receptor levels. In addition, Gα15-mediated coupling of a receptor to PLC can change the cellular response to receptor agonists, indicating that downstream cellular functions can be used to detect receptor activation in screening systems employing a promiscuous G protein. [ABSTRACT FROM AUTHOR]

Abstract Following cell surface receptor activation, the α subunit of the Gq subclass of GTP-binding proteins activates the phosphoinositide signalling pathway. Here we examined the expression and localization of Gq protein α subunits in the adult mouse brain by in situ hybridization and immunohistochemistry. Of the four members of the Gq protein α subunits, Gαq and Gα11 were transcribed predominantly in the brain. The highest transcriptional level of Gαq was observed in cerebellar Purkinje cells (PCs) and hippocampal pyramidal cells, while that of Gα11 was noted in hippocampal pyramidal cells. Antibody against the C-terminal peptide common to Gαq and Gα11 strongly labelled the cerebellar molecular layer and hippocampal neuropil layers. In these regions, immunogold preferentially labelled the cytoplasmic face of postsynaptic cell membrane of PCs and pyramidal cells. Immunoparticles were distributed along the extra-junctional cell membrane of spines, dendrites and somata, but were almost excluded from the junctional membrane. By double immunofluorescence, Gαq/Gα11 was extensively colocalized with metabotropic glutamate receptor mGluR1α in dendritic spines of PCs and with mGluR5 in those of hippocampal pyramidal cells. Together with concentrated localization of mGluR1α and mGluR5 in a peri-junctional annulus on PC and pyramidal cell synapses (Baude et al. 1993, Neuron, 11, 771–787; Luján et al. 1996, Eur. J. Neurosci., 8, 1488–1500), the present molecular-anatomical findings suggest that peri-junctional stimulation of the group I metabotropic glutamate receptors is mediated by Gαq and/or Gα11, leading to the activation of the intracellular effector, phospholipase Cβ. [ABSTRACT FROM AUTHOR]

Details a study which reports that rod outer segment (ROS) membranes from mice lacking the protein regulator RGS9-1 hydrolyze GTP more slowly than ROS membranes from control mice. Effects of the finding on the study of visual perception; Conclusion that RGS9-1 is essential for acceleration of hydrolysis of GTP and for normal recovery of the photoresponse; Outlook.

Heterotrimeric G proteins of the Gq class have been implicated in signaling pathways regulating cardiac growth under physiological and pathological conditions. Knockout mice carrying inactivating mutations in both of the widely expressed Gα q class genes, Gα q and Gα11, demonstrate that at least two active alleles of these genes are required for extrauterine life. Mice carrying only one intact allele [Gα q(­/+);Gα11(­/­) or Gαq(­/­);Gα11(­/+)] died shortly after birth. These mutants showed a high incidence of cardiac malformation. In addition, Gα q(­/­);Gα11(­/+) newborns suffered from craniofacial defects. Mice lacking both Gα q and Gα11 [Gαq(­/­);Gα11(­/­)] died at embryonic day 11 due to cardiomyocyte hypoplasia. These data demonstrate overlap in Gαq and Gα11 gene functions and indicate that the Gq class of G proteins plays a crucial role in cardiac growth and development. [ABSTRACT FROM AUTHOR]

Helical histidine phosphotransferase (HPt) domains play a central role in many aspects of bacterial signal transduction. The 0.98Å resolution crystallographic structure of the amino-terminal HPt domain (P1) from the chemotaxis kinase CheA of Thermotoga maritima reveals a remarkable degree of structural heterogeneity within a four-helix bundle. Two of the four helices have alternate main-chain conformations that differ by a 1.3–1.7Å shift along the bundle axis. These dual conformers were only resolved with atomic resolution diffraction data and their inclusion significantly improved refinement statistics. Neither conformer optimizes packing within the helical core, consistent with their nearly equal refined occupancies. Altered hydrogen bonding within an inter-helical loop may facilitate transition between conformers. Two discrete structural states rather than a continuum of closely related conformations indicates an energetic barrier to conversion between conformers in the crystal at 100K, although many more states are expected in solution at physiological temperatures. Anisotropic atomic thermal B factors within the two conformers indicate modest overall atomic displacement that is largest perpendicular to the helical bundle and not along the direction of apparent motion. Despite the conformational heterogeneity of P1 in the crystal at low temperature, the protein displays high thermal stability in solution (Tm=100°C). Addition of a variable C-terminal region that corresponds to a mobile helix in other CheA structures significantly narrows the temperature width of the unfolding transition and may affect domain dynamics. Helices that compose the kinase recognition site and contain the phospho-accepting His45 do not have alternate conformations. In this region, atomic resolution provides detailed structural parameters for a conserved hydrogen-bonding network that tunes the reactivity of His45. A neighboring glutamate (E67), essential for phosphotransferase activity hydrogen bonds directly to His45 . E67 generates a negative electrostatic surface surrounding the reactive His that is conserved by most CheA kinases, but absent in related phosphotransferase proteins. The P1 conformations that we observe are likely relevant to other helical or coiled-coil proteins and may be important for generating switches in signaling processes. [Copyright &y& Elsevier]