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1.
Biochem Soc Trans ; 41(1): 79-83, 2013 Feb 01.
Article in English | MEDLINE | ID: mdl-23356262

ABSTRACT

Ras GTPases are important regulators of pathways controlling proliferation, differentiation and transformation. Three ubiquitously expressed almost identical Ras genes are not functionally redundant; this has been attributed to their distinctive trafficking and localization profiles. A palmitoylation cycle controls the correct compartmentalization of H-Ras and N-Ras. We review recent data that reveal how this cycle can be regulated by membrane organization to influence the spatiotemporal signalling of Ras.


Subject(s)
Lipoylation , Palmitic Acid/metabolism , Protein Isoforms/metabolism , ras Proteins/metabolism , Amino Acid Sequence , Cell Membrane/metabolism , Molecular Sequence Data , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Transport , Sequence Homology, Amino Acid , Signal Transduction , ras Proteins/chemistry , ras Proteins/genetics
2.
FEBS J ; 276(7): 1800-16, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19243429

ABSTRACT

Ca(2+) regulates a multitude of cellular processes and does so by partitioning its actions in space and time. In this review, we discuss how Ca(2+) responses are constructed from small quantal (elementary) events that have the potential to propagate to produce large pan-cellular responses. We review how Ca(2+) is compartmentalized both physically and functionally, and describe how each organelle has its own distinct Ca(2+)-handling properties. We explain how coordination of the movement of Ca(2+) between organelles is used to shape and hone Ca(2+) signals. Finally, we provide a number of specific examples of where compartmentation and localization of Ca(2+) are crucial to cell function.


Subject(s)
Calcium Signaling/physiology , Calcium/metabolism , Signal Transduction , Animals , Cell Compartmentation/physiology , Cell Nucleus/metabolism , Cytoplasm/metabolism , Humans , Membrane Microdomains/metabolism , Mitochondria/metabolism , Models, Biological
3.
J Cell Sci ; 121(Pt 4): 421-7, 2008 Feb 15.
Article in English | MEDLINE | ID: mdl-18211960

ABSTRACT

RAS isoforms have been proposed to exhibit differing biological outputs due to differences in their relative occupancy of cellular organelles and signalling microdomains. The membrane binding and targeting motifs of RAS are encoded by the C-terminal hypervariable region (HVR), and the precise localisation depends upon interactions between the HVR and the host membrane. Classic studies revealed that all RAS proteins rely on farnesylation and either palmitoylation or a polybasic stretch for stable binding to membranes. We now show that, for N-RAS and Ki-RAS4A, mono-palmitoylation and farnesylation are not sufficient for specifying stable cell-surface localisation. A third motif that is present within the linker domain of all palmitoylated RAS HVRs is necessary for stabilising localisation to the plasma membrane. This motif comprises acidic residues that stabilise palmitoylation and basic amino acids that are likely to interact electrostatically with acidic phospholipids enriched at the cell surface. Importantly, altered localisation is achieved without changes in palmitoylation status. Our data provide a mechanism for distinct HVR membrane interactions controlling subcellular distribution. In the context of the full-length RAS proteins, this is likely to be of crucial importance for controlling signalling output and engagement with different pools of effectors.


Subject(s)
Cell Membrane/metabolism , ras Proteins/metabolism , Amino Acid Sequence , Binding Sites/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HeLa Cells , Humans , Lipoylation , Microscopy, Fluorescence , Molecular Sequence Data , Protein Binding , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Prenylation , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , ras Proteins/genetics
4.
Cell Calcium ; 38(1): 45-51, 2005 Jul.
Article in English | MEDLINE | ID: mdl-15963563

ABSTRACT

Functional assays of inositol 1,4,5-trisphosphate receptors (IP3R) currently use 45Ca2+ release methods, fluorescent Ca2+ indicators within either the ER or cytosol, or electrophysiological analyses of IP3R in the nuclear envelope or artificial bilayers. None of the methods is presently amenable to the rapid, high-throughput quantitative analyses of IP3R function needed to address the structural determinants of IP3R behavior. We use a low-affinity Ca2+ indicator (Mag-fluo-4) to measure free [Ca2+] within the ER of permeabilized DT40 cells expressing only rat type 1 IP(3)R, and establish that the indicator is capable of reliably reporting the Ca(2+) release evoked by IP3. A 96-well fluorescence plate reader equipped for automated fluid additions (FlexStation, Molecular Devices) is used to monitor IP3-evoked Ca2+ release. The method allows quick and economical functional assays of recombinant IP3R in small volumes (< or = 100 microl).


Subject(s)
B-Lymphocytes/metabolism , Calcium Channels/metabolism , Calcium/metabolism , Inositol 1,4,5-Trisphosphate/pharmacology , Receptors, Cytoplasmic and Nuclear/metabolism , Animals , B-Lymphocytes/cytology , B-Lymphocytes/drug effects , Calcium Channels/isolation & purification , Calcium Signaling , Chickens , Cytosol/metabolism , Endoplasmic Reticulum/metabolism , Fluorescent Dyes , Inositol 1,4,5-Trisphosphate Receptors , Receptors, Cytoplasmic and Nuclear/isolation & purification , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism
5.
Mol Membr Biol ; 21(3): 193-205, 2004.
Article in English | MEDLINE | ID: mdl-15204627

ABSTRACT

The plasma membrane consists of a mosaic of functional microdomains facilitating a variety of physiological processes associated with the cell surface. In most cells, the majority of the cell surface is morphologically featureless, leading to difficulties in characterizing its organization and microdomain composition. The reliance on indirect and perturbing techniques has led to vigorous debate concerning the nature and even existence of some microdomains. Recently, increasing technical sophistication has been applied to study cell surface compartmentalization providing evidence for small, short-lived clusters that may be much less than 50 nm in diameter. Lipid rafts and caveolae are cholesterol-dependent, highly ordered microdomains that have received most attention in recent years, yet their precise roles in regulating functions such as cell signalling remain to be determined. Endocytosis of lipid rafts/caveolae follows a clathrin-independent route to both early endosomes and non-classical caveosomes. The observation that a variety of cellular pathogens localize to and internalize with these microdomains provides an additional incentive to characterize the organization, dynamics and functions of these domains.


Subject(s)
Endocytosis/physiology , Membrane Microdomains/chemistry , Membrane Microdomains/physiology , Biological Transport , Cell Compartmentation/physiology , Signal Transduction/physiology , ras Proteins/metabolism
6.
Bioconjug Chem ; 15(2): 278-89, 2004.
Article in English | MEDLINE | ID: mdl-15025523

ABSTRACT

The design and synthesis of dimeric versions of the intracellular signaling molecule d-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] are reported. Ins(1,4,5)P(3) dimers in a range of sizes were constructed by conjugation of a partially protected 2-O-(2-aminoethyl)-Ins(1,4,5)P(3) intermediate with activated oligo- and poly(ethylene glycol) (PEG) tethers, to give benzyl-protected dimers with amide or carbamate linkages. After deprotection, the resulting water-soluble Ins(1,4,5)P(3) dimers were purified by ion-exchange chromatography. The interaction of the Ins(1,4,5)P(3) dimers with tetrameric Ins(1,4,5)P(3) receptors was explored, using equilibrium [(3)H]Ins(1,4,5)P(3)-binding to membranes from cerebellum, and (45)Ca(2+)-release from permeabilized hepatocytes. The results showed that dimers, even when they incorporate large PEG tethers, interact potently with Ins(1,4,5)P(3) receptors, and that the shorter dimers are more potent than Ins(1,4,5)P(3) itself. A very small dimer, consisting of two Ins(1,4,5)P(3) motifs joined by a short N,N'-diethylurea spacer, was synthesized. Preliminary studies of (45)Ca(2+) release from the intracellular stores of permeabilized hepatocytes showed this shortest dimer to be almost as potent as adenophostin A, the most potent Ins(1,4,5)P(3) receptor ligand known. Possible interpretations of this result are considered in relation to the recently disclosed X-ray crystal structure of the type 1 Ins(1,4,5)P(3) receptor core binding domain.


Subject(s)
Calcium Channels/metabolism , Drug Design , Inositol 1,4,5-Trisphosphate/analogs & derivatives , Inositol 1,4,5-Trisphosphate/chemical synthesis , Inositol 1,4,5-Trisphosphate/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Animals , Binding, Competitive , Cell Membrane/metabolism , Cell Membrane Permeability , Cerebellum/metabolism , Dimerization , Hepatocytes/metabolism , Inositol 1,4,5-Trisphosphate Receptors , Mice , Protein Binding , Rats
7.
J Med Chem ; 46(23): 4860-71, 2003 Nov 06.
Article in English | MEDLINE | ID: mdl-14584937

ABSTRACT

The synthesis of a series of adenophostin A analogues modified at C-6 and C-2 of adenine is described. The target compounds were synthesized by a convergent route involving a modified Vorbrüggen condensation of either 6-chloropurine or 2,6-dichloropurine with a protected disaccharide, yielding two versatile intermediates capable of undergoing substitution with a range of nucleophiles. The new analogues showed a range of abilities to mobilize Ca(2+) from the intracellular stores of permeabilized hepatocytes and are among the first totally synthetic compounds to approach the activity of adenophostin A. In agreement with the biological results, docking studies of adenophostin A using the recently reported X-ray crystal structure of the type 1 Ins(1,4,5)P(3) receptor binding core suggested that, in likely binding modes of adenophostin A, the area around N(6) may be relatively open, identifying this region of the adenophostin A molecule as a promising target for further elaboration. The docking results also point to specific interactions involving residues within the binding domain of the Ins(1,4,5)P(3) receptor that may be involved in the molecular recognition of the adenophostins.


Subject(s)
Adenosine/analogs & derivatives , Adenosine/chemical synthesis , Calcium Channels/chemistry , Calcium/metabolism , Purines/chemistry , Receptors, Cytoplasmic and Nuclear/chemistry , Adenosine/chemistry , Adenosine/metabolism , Adenosine/pharmacology , Animals , Binding Sites , Calcium Channels/metabolism , Hepatocytes/drug effects , Hepatocytes/metabolism , In Vitro Techniques , Inositol 1,4,5-Trisphosphate Receptors , Models, Molecular , Molecular Mimicry , Rats , Receptors, Cytoplasmic and Nuclear/metabolism , Structure-Activity Relationship
8.
Pflugers Arch ; 445(5): 614-21, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12634934

ABSTRACT

We describe the actions of 2,3-butanedione monoxime (BDM) on calcium responses in secretory cells. Our studies were prompted by the widespread use of BDM as a myosin-ATPase inhibitor. Application of 10 mM BDM almost completely inhibited agonist-evoked amylase secretion from mouse pancreatic acinar cells. This action might be interpreted as indicating a role for myosin in secretion. However, BDM alone elicited a calcium response in single cells and this calcium signal was sufficient to activate calcium-dependent chloride currents. Furthermore, in some cases, BDM potentiated agonist-evoked calcium signals but almost always blocked agonist-evoked calcium oscillations. These effects of BDM were not due to an action on calcium influx pathways but rather to direct effects on IP(3)-sensitive stores. We conclude that BDM cannot be used for unequivocal identification of the involvement of myosin motors in a cellular response. Further, our evidence suggests that BDM can act directly to modify the opening of IP(3) receptors.


Subject(s)
Calcium/metabolism , Diacetyl/analogs & derivatives , Diacetyl/pharmacology , Enzyme Inhibitors/pharmacology , Inositol 1,4,5-Trisphosphate/physiology , Pancreas/metabolism , Acetylcholine/pharmacology , Animals , Animals, Outbred Strains , Calcium/physiology , Electrophysiology , Intracellular Membranes/metabolism , Male , Mice , Myosins/antagonists & inhibitors , Oscillometry , Osmolar Concentration , Pancreas/cytology , Pancreas/drug effects , Patch-Clamp Techniques
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