Spatiotemporal regulation of PKC via interactions with AKAP7 isoforms

2012 ◽  
Vol 446 (2) ◽  
pp. 301-309 ◽  
Author(s):  
John M. Redden ◽  
Andrew V. Le ◽  
Arpita Singh ◽  
Kyle Federkiewicz ◽  
Samantha Smith ◽  
...  
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Scaffolding Protein ◽  
Scaffolding Proteins ◽  
Protein Biochemistry ◽  
Spatiotemporal Regulation ◽  
Anchoring Protein ◽  
Integral Role ◽  
Pkc Protein Kinase C ◽  
And Function

The regulation of kinases by scaffolding proteins greatly contributes to the fidelity of signal transduction. In the present study, we explored an interaction between the ubiquitous enzyme PKC (protein kinase C) and the scaffolding protein AKAP7 (A-kinase-anchoring protein 7). Using protein biochemistry and surface plasmon resonance approaches, we demonstrate that both AKAP7γ and AKAP7α are capable of high-affinity interactions with multiple isoenzymes of PKC. Furthermore, this interaction is achieved via multi-site binding on both proteins. FRET (fluorescence resonance energy transfer) analysis using a PKC activity reporter suggests that anchoring of the kinase within AKAP7 complexes enhances the phosphorylation of substrate proteins. Finally, we determined using FRAP (fluorescence recovery after photobleaching) and virtual modelling that AKAP7 restricts the mobility of PKC within cells by tethering it to subcellular compartments. Collectively, the results of the present study suggests that AKAP7 could play an integral role in dictating PKC localization and function in tissues where the two proteins are co-expressed.

scholarly journals Discs large 1 controls daughter-cell polarity after cytokinesis in vertebrate morphogenesis

2018 ◽  
Vol 115 (46) ◽  
pp. E10859-E10868 ◽  
Author(s):  
Yuwei Li ◽  
Jason A. Junge ◽  
Cosimo Arnesano ◽  
Garrett G. Gross ◽  
Jeffrey H. Miner ◽  
...  
Keyword(s):  
Cell Polarity ◽  
Protein Interactions ◽  
Protein Function ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Explant Culture ◽  
Scaffolding Protein ◽  
Fluorescence Lifetime Imaging ◽  
Discs Large

Vertebrate embryogenesis and organogenesis are driven by cell biological processes, ranging from mitosis and migration to changes in cell size and polarity, but their control and causal relationships are not fully defined. Here, we use the developing limb skeleton to better define the relationships between mitosis and cell polarity. We combine protein-tagging and -perturbation reagents with advanced in vivo imaging to assess the role of Discs large 1 (Dlg1), a membrane-associated scaffolding protein, in mediating the spatiotemporal relationship between cytokinesis and cell polarity. Our results reveal that Dlg1 is enriched at the midbody during cytokinesis and that its multimerization is essential for the normal polarity of daughter cells. Defects in this process alter tissue dimensions without impacting other cellular processes. Our results extend the conventional view that division orientation is established at metaphase and anaphase and suggest that multiple mechanisms act at distinct phases of the cell cycle to transmit cell polarity. The approach employed can be used in other systems, as it offers a robust means to follow and to eliminate protein function and extends the Phasor approach for studying in vivo protein interactions by frequency-domain fluorescence lifetime imaging microscopy of Förster resonance energy transfer (FLIM-FRET) to organotypic explant culture.

scholarly journals Lipid Bilayer Composition Affects Transmembrane Protein Orientation and Function

2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
Katie D. Hickey ◽  
Mary M. Buhr
Keyword(s):  
Lipid Bilayer ◽  
Lipid Composition ◽  
Protein Function ◽  
Transmembrane Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Outer Leaflet ◽  
Lipid Environment ◽  
Protein Incorporation ◽  
And Function

Sperm membranes change in structure and composition upon ejaculation to undergo capacitation, a molecular transformation which enables spermatozoa to undergo the acrosome reaction and be capable of fertilization. Changes to the membrane environment including lipid composition, specifically lipid microdomains, may be responsible for enabling capacitation. To study the effect of lipid environment on proteins, liposomes were created using lipids extracted from bull sperm membranes, with or without a protein (Na+K+-ATPase or -amylase). Protein incorporation, function, and orientation were determined. Fluorescence resonance energy transfer (FRET) confirmed protein inclusion in the lipid bilayer, and protein function was confirmed using a colourometric assay of phosphate production from ATP cleavage. In the native lipid liposomes, ATPase was oriented with the subunit facing the outer leaflet, while changing the lipid composition to 50% native lipids and 50% exogenous lipids significantly altered this orientation of Na+K+-ATPase within the membranes.

scholarly journals Class Frizzled GPCRs (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

2019 ◽  
Vol 2019 (4) ◽  
Author(s):  
Elisa Arthofer ◽  
Jacomijn Dijksterhuis ◽  
Belma Hot ◽  
Paweł Kozielewicz ◽  
Matthias Lauth ◽  
...  
Keyword(s):  
G Proteins ◽  
Conformational Changes ◽  
Hedgehog Signaling ◽  
Cell Signalling ◽  
Resonance Energy Transfer ◽  
Rho Kinase ◽  
Density Lipoprotein ◽  
Resonance Energy ◽  
Scaffolding Protein ◽  
Heterotrimeric G Proteins

Receptors of the Class Frizzled (FZD, nomenclature as agreed by the NC-IUPHAR subcommittee on the Class Frizzled GPCRs [156]), are GPCRs originally identified in Drosophila [17], which are highly conserved across species. While SMO shows structural resemblance to the 10 FZDs, it is functionally separated as it mediates effects in the Hedgehog signaling pathway [156]. FZDs are activated by WNTs, which are cysteine-rich lipoglycoproteins with fundamental functions in ontogeny and tissue homeostasis. FZD signalling was initially divided into two pathways, being either dependent on the accumulation of the transcription regulator β-catenin or being β-catenin-independent (often referred to as canonical vs. non-canonical WNT/FZD signalling, respectively). WNT stimulation of FZDs can, in cooperation with the low density lipoprotein receptors LRP5 (O75197) and LRP6 (O75581), lead to the inhibition of a constitutively active destruction complex, which results in the accumulation of β-catenin and subsequently its translocation to the nucleus. β-Catenin, in turn, modifies gene transcription by interacting with TCF/LEF transcription factors. β-Catenin-independent FZD signalling is far more complex with regard to the diversity of the activated pathways. WNT/FZD signalling can lead to the activation of heterotrimeric G proteins [28, 159, 135], the elevation of intracellular calcium [164], activation of cGMP-specific PDE6 [2] and elevation of cAMP as well as RAC-1, JNK, Rho and Rho kinase signalling [48]. Novel resonance energy transfer-based tools have allowed the study of the GPCR-like nature of FZDs in greater detail. Upon ligand stimulation, FZDs undergo conformational changes and signal via heterotrimeric G proteins [213, 214]. Furthermore, the phosphoprotein Dishevelled constitutes a key player in WNT/FZD signalling. Importantly, FZDs exist in at least two distinct conformational states that regulate the pathway selection [214]. As with other GPCRs, members of the Frizzled family are functionally dependent on the arrestin scaffolding protein for internalization [19], as well as for β-catenin-dependent [12] and -independent [80, 13] signalling. The pattern of cell signalling is complicated by the presence of additional ligands, which can enhance or inhibit FZD signalling (secreted Frizzled-related proteins (sFRP), Wnt-inhibitory factor (WIF), sclerostin or Dickkopf (DKK)), as well as modulatory (co)-receptors with Ryk, ROR1, ROR2 and Kremen, which may also function as independent signalling proteins.

scholarly journals Dynamic remodeling of scaffold interactions in dendritic spines controls synaptic excitability

2012 ◽  
Vol 198 (2) ◽  
pp. 251-263 ◽  
Author(s):  
Enora Moutin ◽  
Fabrice Raynaud ◽  
Jonathan Roger ◽  
Emilie Pellegrino ◽  
Vincent Homburger ◽  
...  
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Membrane Receptors ◽  
Physical Interaction ◽  
Scaffolding Proteins ◽  
Cellular Functions ◽  
Electrophysiological Recordings ◽  
Living Neurons ◽  
Activity Dependent

Scaffolding proteins interact with membrane receptors to control signaling pathways and cellular functions. However, the dynamics and specific roles of interactions between different components of scaffold complexes are poorly understood because of the dearth of methods available to monitor binding interactions. Using a unique combination of single-cell bioluminescence resonance energy transfer imaging in living neurons and electrophysiological recordings, in this paper, we depict the role of glutamate receptor scaffold complex remodeling in space and time to control synaptic transmission. Despite a broad colocalization of the proteins in neurons, we show that spine-confined assembly/disassembly of this scaffold complex, physiologically triggered by sustained activation of synaptic NMDA (N-methyl-d-aspartate) receptors, induces physical association between ionotropic (NMDA) and metabotropic (mGlu5a) synaptic glutamate receptors. This physical interaction results in an mGlu5a receptor–mediated inhibition of NMDA currents, providing an activity-dependent negative feedback loop on NMDA receptor activity. Such protein scaffold remodeling represents a form of homeostatic control of synaptic excitability.

scholarly journals Regulatory γ1 subunits defy symmetry in functional modulation of BK channels

2018 ◽  
Vol 115 (40) ◽  
pp. 9923-9928 ◽  
Author(s):  
Vivian Gonzalez-Perez ◽  
Manu Ben Johny ◽  
Xiao-Ming Xia ◽  
Christopher J. Lingle
Keyword(s):  
Single Channel ◽  
Regulatory Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bk Channels ◽  
Bk Channel ◽  
Single Type ◽  
Regulatory Subunits ◽  
And Function

Structural symmetry is a hallmark of homomeric ion channels. Nonobligatory regulatory proteins can also critically define the precise functional role of such channels. For instance, the pore-forming subunit of the large conductance voltage and calcium-activated potassium (BK, Slo1, or KCa1.1) channels encoded by a single KCa1.1 gene assembles in a fourfold symmetric fashion. Functional diversity arises from two families of regulatory subunits, β and γ, which help define the range of voltages over which BK channels in a given cell are activated, thereby defining physiological roles. A BK channel can contain zero to four β subunits per channel, with each β subunit incrementally influencing channel gating behavior, consistent with symmetry expectations. In contrast, a γ1 subunit (or single type of γ1 subunit complex) produces a functionally all-or-none effect, but the underlying stoichiometry of γ1 assembly and function remains unknown. Here we utilize two distinct and independent methods, a Forster resonance energy transfer-based optical approach and a functional reporter in single-channel recordings, to reveal that a BK channel can contain up to four γ1 subunits, but a single γ1 subunit suffices to induce the full gating shift. This requires that the asymmetric association of a single regulatory protein can act in a highly concerted fashion to allosterically influence conformational equilibria in an otherwise symmetric K+channel.

scholarly journals Imaging kinase–AKAP79–phosphatase scaffold complexes at the plasma membrane in living cells using FRET microscopy

2002 ◽  
Vol 160 (1) ◽  
pp. 101-112 ◽  
Author(s):  
Seth F. Oliveria ◽  
Lisa L. Gomez ◽  
Mark L. Dell'Acqua
Keyword(s):  
Signal Transduction ◽  
Glutamate Receptors ◽  
Direct Evidence ◽  
Protein A ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Adaptor Proteins ◽  
Living Cells ◽  
Intact Cells ◽  
Anchoring Protein

Scaffold, anchoring, and adaptor proteins coordinate the assembly and localization of signaling complexes providing efficiency and specificity in signal transduction. The PKA, PKC, and protein phosphatase-2B/calcineurin (CaN) scaffold protein A–kinase anchoring protein (AKAP) 79 is localized to excitatory neuronal synapses where it is recruited to glutamate receptors by interactions with membrane-associated guanylate kinase (MAGUK) scaffold proteins. Anchored PKA and CaN in these complexes could have important functions in regulating glutamate receptors in synaptic plasticity. However, direct evidence for the assembly of complexes containing PKA, CaN, AKAP79, and MAGUKs in intact cells has not been available. In this report, we use immunofluorescence and fluorescence resonance energy transfer (FRET) microscopy to demonstrate membrane cytoskeleton–localized assembly of this complex. Using FRET, we directly observed binding of CaN catalytic A subunit (CaNA) and PKA-RII subunits to membrane-targeted AKAP79. We also detected FRET between CaNA and PKA-RII bound simultaneously to AKAP79 within 50 Å of each other, thus providing the first direct evidence of a ternary kinase–scaffold–phosphatase complex in living cells. This finding of AKAP-mediated PKA and CaN colocalization on a nanometer scale gives new appreciation to the level of compartmentalized signal transduction possible within scaffolds. Finally, we demonstrated AKAP79-regulated membrane localization of the MAGUK synapse-associated protein 97 (SAP97), suggesting that AKAP79 functions to organize even larger signaling complexes.

scholarly journals Muscarinic receptors stimulate AC2 by novel phosphorylation sites, whereas Gβγ subunits exert opposing effects depending on the G-protein source

2012 ◽  
Vol 447 (3) ◽  
pp. 393-405 ◽  
Author(s):  
Jia X. Shen ◽  
Sebastian Wachten ◽  
Michelle L. Halls ◽  
Katy L. Everett ◽  
Dermot M. F. Cooper
Keyword(s):  
Muscarinic Receptor ◽  
Somatostatin Receptors ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Phosphorylation Sites ◽  
Canonical Pathways ◽  
Pkc Protein Kinase C ◽  
Opposite Manner ◽  
Opposing Effects

Direct phosphorylation of AC2 (adenylyl cyclase 2) by PKC (protein kinase C) affords an opportunity for AC2 to integrate signals from non-canonical pathways to produce the second messenger, cyclic AMP. The present study shows that stimulation of AC2 by pharmacological activation of PKC or muscarinic receptor activation is primarily the result of phosphorylation of Ser490 and Ser543, as opposed to the previously proposed Thr1057. A double phosphorylation-deficient mutant (S490/543A) of AC2 was insensitive to PMA (phorbol myristic acid) and CCh (carbachol) stimulation, whereas a double phosphomimetic mutant (S490/543D) mimicked the activity of PKC-activated AC2. Putative Gβγ-interacting sites are in the immediate environment of these PKC phosphorylation sites (Ser490 and Ser543) that are located within the C1b domain of AC2, suggesting a significant regulatory importance of this domain. Consequently, we examined the effect of both Gq-coupled muscarinic and Gi-coupled somatostatin receptors. Employing pharmacological and FRET (fluorescence resonance energy transfer)-based real-time single cell imaging approaches, we found that Gβγ released from the Gq-coupled muscarinic receptor or Gi-coupled somatostatin receptors exert inhibitory or stimulatory effects respectively. These results underline the sophisticated regulatory capacities of AC2, in not only being subject to regulation by PKC, but also and in an opposite manner to Gβγ subunits, depending on their source.

scholarly journals Methods used to study the oligomeric structure of G-protein-coupled receptors

2017 ◽  
Vol 37 (2) ◽  
Author(s):  
Hui Guo ◽  
Su An ◽  
Richard Ward ◽  
Yang Yang ◽  
Ying Liu ◽  
...  
Keyword(s):  
Energy Transfer ◽  
G Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
G Protein Coupled Receptors ◽  
Experimental Techniques ◽  
Distribution Analysis ◽  
Wide Range ◽  
And Function ◽  
G Protein Coupled

G-protein-coupled receptors (GPCRs), which constitute the largest family of cell surface receptors, were originally thought to function as monomers, but are now recognized as being able to act in a wide range of oligomeric states and indeed, it is known that the oligomerization state of a GPCR can modulate its pharmacology and function. A number of experimental techniques have been devised to study GPCR oligomerization including those based upon traditional biochemistry such as blue-native PAGE (BN-PAGE), co-immunoprecipitation (Co-IP) and protein-fragment complementation assays (PCAs), those based upon resonance energy transfer, FRET, time-resolved FRET (TR-FRET), FRET spectrometry and bioluminescence resonance energy transfer (BRET). Those based upon microscopy such as FRAP, total internal reflection fluorescence microscopy (TIRFM), spatial intensity distribution analysis (SpIDA) and various single molecule imaging techniques. Finally with the solution of a growing number of crystal structures, X-ray crystallography must be acknowledged as an important source of discovery in this field. A different, but in many ways complementary approach to the use of more traditional experimental techniques, are those involving computational methods that possess obvious merit in the study of the dynamics of oligomer formation and function. Here, we summarize the latest developments that have been made in the methods used to study GPCR oligomerization and give an overview of their application.

scholarly journals Functional Interaction between the N and C Termini of NhaD Antiporters from Halomonas sp. Strain Y2

2017 ◽  
Vol 199 (16) ◽  
Author(s):  
Yiwei Meng ◽  
Zhou Yang ◽  
Bin Cheng ◽  
Xinyu Nie ◽  
Shannan Li ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Ph Regulation ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Limited Information ◽  
High Sequence Identity ◽  
Ph Profile ◽  
Content Type ◽  
Ion Translocation ◽  
And Function

ABSTRACT Two NhaD-type antiporters, NhaD1 and NhaD2, from the halotolerant and alkaliphilic Halomonas sp. strain Y2, exhibit different physiological functions in regard to Na+ and Li+ resistance, although they share high sequence identity. In the present study, the truncation of an additional 4 C-terminal residues from NhaD2 or an exchange of 39 N-terminal residues between these proteins resulted in the complete loss of antiporter activity. Interestingly, combining 39 N-terminal residues and 7 C-terminal residues of NhaD2 (N39D2-C7) partially recovered the activity for Na+ and Li+ expulsion, as well as complementary growth following exposure to 300 mM Na+ and 100 mM Li+ stress. The recovered activity of chimera N39D2-C7 indicated that the N and C termini are structurally dependent on each other and function synergistically. Furthermore, fluorescence resonance energy transfer (FRET) analysis suggested that the N and C termini are relatively close in proximity which may account for their synergistic function in ion translocation. In the N-terminal region of N39D2-C7, the replacement of Glu38 with Pro abolished the recovered complementary and transport activities. In addition, this amino acid substitution in NhaD2 resulted in a drastically decreased complementation ability in Escherichia coli KNabc (level identical to that of NhaD1), as well as decreased activity and an altered pH profile. IMPORTANCE Limited information on NhaD antiporters supports speculation that these antiporters are important for resistance to high salinity and alkalinity. Moreover, only a few functional residues have been identified in NhaD antiporters, and there is limited literature on the molecular mechanisms of NhaD antiporter activity. The altered antiporter abilities of chimeras and mutants in this study implicate the functions of the N and C termini, especially Glu38, in pH regulation and ion translocation, and, most importantly, the essential roles of this negatively charged residue in maintaining the physiological function of NhaD2. These findings further our understanding of the molecular mechanism of NhaD antiporters for ion transport.

scholarly journals Multiplexed, bioorthogonal labeling of multicomponent, biomolecular complexes using genomically encoded, non-canonical amino acids

2019 ◽  
Author(s):  
Bijoy J. Desai ◽  
Ruben L. Gonzalez
Keyword(s):  
Amino Acids ◽  
Structural Biology ◽  
Structural Dynamics ◽  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Functional Studies ◽  
Bioorthogonal Labeling ◽  
Biomolecular Complexes ◽  
And Function

Stunning advances in the structural biology of multicomponent biomolecular complexes (MBCs) have ushered in an era of intense, structure-guided mechanistic and functional studies of these complexes. Nonetheless, existing methods to site-specifically conjugate MBCs with biochemical and biophysical labels are notoriously impracticable and/or significantly perturb MBC assembly and function. To overcome these limitations, we have developed a general, multiplexed method in which we genomically encode non-canonical amino acids (ncAAs) into multiple, structure-informed, individual sites within a target MBC; select for ncAA-containing MBC variants that assemble and function like the wildtype MBC; and site-specifically conjugate biochemical or biophysical labels to these ncAAs. As a proof-of-principle, we have used this method to generate unique single-molecule fluorescence resonance energy transfer (smFRET) signals reporting on ribosome structural dynamics that have thus far remained inaccessible to smFRET studies of translation.

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