PIP2 binds vinculin and directs its oligomerization, which promotes proper focal adhesion structure and function.

SUMMARY The G protein alpha subunit o (Gαo) is one of the most abundant proteins in the nervous system, and pathogenic mutations in its gene ( GNAO1 ) cause movement disorder. However, the function of Gαo is ill defined mechanistically. Here, we show that Gαo dictates neuromodulatory responsiveness of striatal neurons and is required for movement control. Using in vivo optical sensors and enzymatic assays, we determine that Gαo provides a separate transduction channel that modulates coupling of both inhibitory and stimulatory dopamine receptors to the cyclic AMP (cAMP)-generating enzyme adenylyl cyclase. Through a combination of cell-based assays and rodent models, we demonstrate that GNAO1 -associated mutations alter Gαo function in a neuron-type-specific fashion via a combination of a dominant-negative and loss-of-function mechanisms. Overall, our findings suggest that Gαo and its pathological variants function in specific circuits to regulate neuromodulatory signals essential for executing motor programs.

Functional characterization of the GPCR interactome has been focused predominantly on intracellular interactions, yet GPCRs are increasingly found in complex with extracellular proteins. Extracellular leucine-rich repeat fibronectin type III domain containing 1 (ELFN1) was recently reported to physically anchor mGluR6 and mGluR7 across retinal and hippocampal synapses, respectively; however, the consequence of transsynaptic interactions on properties and pharmacology of these receptors are unknown. In the current study, we explore the effects of ELFN1 on mGluR signaling and pharmacology. First, we established the binding specificity of ELFN1 and found it to be recruited selectively to all group III mGluRs (mGluR4, mGluR6, mGluR7, and mGluR8), but not other mGluR species. Using site-directed mutagenesis we mapped binding determinants of this interaction to two distinct sites on the ELFN1 ectodomain. To evaluate functional aspects of the interaction, we developed a transcellular signaling assay in reconstituted HEK293 cells which monitors changes in mGluR activity in one cell following its exposure to separate ELFN1-containing cells. Using this platform, we found that ELFN1 acts as an allosteric modulator of class III mGluR activity in suppressing cAMP accumulation: altering both agonist-induced and constitutive receptor activity. Using bioluminescence resonance energy transfer-based real-time kinetic assays, we established that ELFN1 alters the ability of mGluRs to activate G proteins. Our findings demonstrate that core properties of class III mGluRs can be altered via extracellular interactions with ELFN1 which serves as a transsynaptic allosteric modulator for these receptors. Furthermore, our unique assay platform opens avenues for exploring transcellular/transsynaptic pharmacology of other GPCR transcomplexes.

Global developmental delay (GDD), often accompanied by intellectual disability, seizures and other features is a severe, clinically and genetically highly heterogeneous childhood-onset disorder. In cases where genetic causes have been identified, de novo mutations in neuronally expressed genes are a common scenario. These mutations can be best identified by exome sequencing of parent-offspring trios. De novo mutations in the guanine nucleotide-binding protein, beta 1 (GNB1) gene, encoding the Gβ1 subunit of heterotrimeric G proteins, have recently been identified as a novel genetic cause of GDD. Using exome sequencing, we identified 14 different novel variants (2 splice site, 2 frameshift and 10 missense changes) in GNB1 in 16 pediatric patients. One mutation (R96L) was recurrently found in three ethnically diverse families with an autosomal dominant mode of inheritance. Ten variants occurred de novo in the patients. Missense changes were functionally tested for their pathogenicity by assaying the impact on complex formation with Gγ and resultant mutant Gβγ with Gα. Signaling properties of G protein complexes carrying mutant Gβ1 subunits were further analyzed by their ability to couple to dopamine D1R receptors by real-time bioluminescence resonance energy transfer (BRET) assays. These studies revealed altered functionality of the missense mutations R52G, G64V, A92T, P94S, P96L, A106T and D118G but not for L30F, H91R and K337Q. In conclusion, we demonstrate a pathogenic role of de novo and autosomal dominant mutations in GNB1 as a cause of GDD and provide insights how perturbation in heterotrimeric G protein function contributes to the disease.

The plant genome contains a large number of sequences that encode catalytically inactive chitinases referred to as chitinase-like proteins (CLPs). Although CLPs share high sequence and structural homology with chitinases of glycosyl hydrolase 18 (TIM barrel domain) and 19 families, they may lack the binding/catalytic activity. Molecular genetic analysis revealed that gene duplication events followed by mutation in the existing chitinase gene have resulted in the loss of activity. The evidences show that adaptive functional diversification of the CLPs has been achieved through alterations in the flexible regions than in the rigid structural elements. The CLPs plays an important role in the defense response against pathogenic attack, biotic and abiotic stress. They are also involved in the growth and developmental processes of plants. Since the physiological roles of CLPs are similar to chitinase, such mutations have led to plurifunctional enzymes. The biochemical and structural characterization of the CLPs is essential for understanding their roles and to develop potential utility in biotechnological industries. This review sheds light on the structure-function evolution of CLPs from chitinases.

Not your typical GPCR Among the large family of G protein–coupled receptors (GPCRs) are many orphans, so called because their signaling reactions remain poorly understood. Among these is GPR158 which is highly expressed in the nervous system and implicated in processes from cognition to memory to mood. Patil et al . determined a high-resolution structure of GPR158 alone and bound to a regulator of G protein signaling (RGS) complex. GPR158 has an unusual dimerization mode with an extensive interaction interface that locks it in a conformation that likely prevents G protein activation. RGS binds to the homodimer at a site that substantially overlaps the surface that binds G proteins, again preventing canonical G protein signaling. Binding of a ligand to the extracellular domain may regulate signaling through the RGS complex. —VV

BackgroundNeuropsychiatric disorders are common forms of disability in humans. Despite recent progress in deciphering the genetics of these disorders, their phenotypic complexity continues to be a major challenge. Mendelian neuropsychiatric disorders are rare but their study has the potential to unravel novel mechanisms that are relevant to their complex counterparts.ResultsIn an extended consanguineous family, we identified a novel neuropsychiatric phenotype characterized by severe speech impairment, variable expressivity of attention deficit hyperactivity disorder (ADHD), and motor delay. We identified the disease locus through linkage analysis on 15q21.2, and exome sequencing revealed a novel missense variant in GNB5. GNB5 encodes an atypical β subunit of the heterotrimeric GTP-binding proteins (Gβ5). Gβ5 is enriched in the central nervous system where it forms constitutive complexes with members of the regulator of G protein signaling family of proteins to modulate neurotransmitter signaling that affects a number of neurobehavioral outcomes. Here, we show that the S81L mutant form of Gβ5 has significantly impaired activity in terminating responses that are elicited by dopamine.ConclusionsWe demonstrate that these deficits originate from the impaired expression of the mutant Gβ5 protein, resulting in the decreased ability to stabilize regulator of G protein signaling complexes. Our data suggest that this novel neuropsychiatric phenotype is the human equivalent of Gnb5 deficiency in mice, which manifest motor deficits and hyperactivity, and highlight a critical role of Gβ5 in normal behavior as well as language and motor development in humans.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-1061-6) contains supplementary material, which is available to authorized users.

Expression of KdpFABC, a K+ pump that restores osmotic balance, is controlled by binding of the response regulator KdpE to a specific DNA sequence (kdpFABCBS) via the winged helix-turn-helix type DNA binding domain (KdpEDBD). Exploration of E. coli KdpEDBD and kdpFABCBS interaction resulted in the identification of two conserved, AT-rich 6 bp direct repeats that form half-sites. Despite binding to these half-sites, KdpEDBD was incapable of promoting gene expression in vivo. Structure-function studies guided by our 2.5 Å X-ray structure of KdpEDBD revealed the importance of residues R193 and R200 in the α-8 DNA recognition helix and T215 in the wing region for DNA binding. Mutation of these residues renders KdpE incapable of inducing expression of the kdpFABC operon. Detailed biophysical analysis of interactions using analytical ultracentrifugation revealed a 2∶1 stoichiometry of protein to DNA with dissociation constants of 200±100 and 350±100 nM at half-sites. Inactivation of one half-site does not influence binding at the other, indicating that KdpEDBD binds independently to the half-sites with approximately equal affinity and no discernable cooperativity. To our knowledge, these data are the first to describe in quantitative terms the binding at half-sites under equilibrium conditions for a member of the ubiquitous OmpR/PhoB family of proteins.