Regulation of the Subcellular Localization of the G-protein Subunit Regulator GPSM3 through Direct Association with 14-3-3 Protein.
ABSTRACT G-protein signaling modulator-3 (GPSM3), also known as G18 or AGS4, is a member of the Gα(i/o)-Loco (GoLoco) motif containing proteins. GPSM3 acts through its two GoLoco motifs to exert GDP dissociation inhibitor activity over Gα(i) subunits; recently revealed is the existence of an additional regulatory site within GPSM3 directed toward monomeric Gβ subunits during their biosynthesis. Here, using in silico and proteomic approaches, we have found that GPSM3 also interacts directly with numerous members of the 14-3-3 protein family. This interaction is dependent on GPSM3 phosphorylation, creating a mode II consensus 14-3-3 binding site. 14-3-3 binding to the N-terminal disordered region of GPSM3 confers stabilization from protein degradation. The complex of GPSM3 and 14-3-3 is exclusively cytoplasmic, and both moieties mutually control their exclusion from the nucleus. Phosphorylation of GPSM3 by a proline-directed serine/threonine kinase and the resultant association of 14-3-3 is the first description of post-translational regulation of GPSM3 subcellular localization, a process that likely regulates important spatio-temporal aspects of G-protein-coupled receptor signaling modulation by GPSM3.
- SourceAvailable from: ncbi.nlm.nih.gov[show abstract] [hide abstract]
ABSTRACT: Protein interactions are a fundamental mechanism for the generation of biological regulatory specificity. The study of protein interactions in living cells is of particular significance because the interactions that occur in a particular cell depend on the full complement of proteins present in the cell and the external stimuli that influence the cell. Bimolecular fluorescence complementation (BiFC) analysis enables direct visualization of protein interactions in living cells. The BiFC assay is based on the association between two nonfluorescent fragments of a fluorescent protein when they are brought in proximity to each other by an interaction between proteins fused to the fragments. Numerous protein interactions have been visualized using the BiFC assay in many different cell types and organisms. The BiFC assay is technically straightforward and can be performed using standard molecular biology and cell culture reagents and a regular fluorescence microscope or flow cytometer.Annual Review of Biophysics 02/2008; 37:465-87. · 12.63 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: G protein-coupled receptors represent the largest family of membrane receptors that instigate signalling through nucleotide exchange on heterotrimeric G proteins. Nucleotide exchange, or more precisely, GDP dissociation from the G protein α-subunit, is the key step towards G protein activation and initiation of downstream signalling cascades. Despite a wealth of biochemical and biophysical studies on inactive and active conformations of several heterotrimeric G proteins, the molecular underpinnings of G protein activation remain elusive. To characterize this mechanism, we applied peptide amide hydrogen-deuterium exchange mass spectrometry to probe changes in the structure of the heterotrimeric bovine G protein, Gs (the stimulatory G protein for adenylyl cyclase) on formation of a complex with agonist-bound human β(2) adrenergic receptor (β(2)AR). Here we report structural links between the receptor-binding surface and the nucleotide-binding pocket of Gs that undergo higher levels of hydrogen-deuterium exchange than would be predicted from the crystal structure of the β(2)AR-Gs complex. Together with X-ray crystallographic and electron microscopic data of the β(2)AR-Gs complex (from refs 2, 3), we provide a rationale for a mechanism of nucleotide exchange, whereby the receptor perturbs the structure of the amino-terminal region of the α-subunit of Gs and consequently alters the 'P-loop' that binds the β-phosphate in GDP. As with the Ras family of small-molecular-weight G proteins, P-loop stabilization and β-phosphate coordination are key determinants of GDP (and GTP) binding affinity.Nature 01/2011; 477(7366):611-5. · 38.60 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: A major challenge in the proteomics and structural genomics era is to predict protein structure and function, including identification of those proteins that are partially or wholly unstructured. Non-globular sequence segments often contain short linear peptide motifs (e.g. SH3-binding sites) which are important for protein function. We present here a new tool for discovery of such unstructured, or disordered regions within proteins. GlobPlot (http://globplot.embl.de) is a web service that allows the user to plot the tendency within the query protein for order/globularity and disorder. We show examples with known proteins where it successfully identifies inter-domain segments containing linear motifs, and also apparently ordered regions that do not contain any recognised domain. GlobPlot may be useful in domain hunting efforts. The plots indicate that instances of known domains may often contain additional N- or C-terminal segments that appear ordered. Thus GlobPlot may be of use in the design of constructs corresponding to globular proteins, as needed for many biochemical studies, particularly structural biology. GlobPlot has a pipeline interface--GlobPipe--for the advanced user to do whole proteome analysis. GlobPlot can also be used as a generic infrastructure package for graphical displaying of any possible propensity.Nucleic Acids Research 08/2003; 31(13):3701-8. · 8.28 Impact Factor