The role of palmitoylation in regulating Ras localization and function.

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.

Compartmentalized signalling: Ca2+ compartments, microdomains and the many facets of Ca2+ signalling.

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.

Palmitoylation and localisation of RAS isoforms are modulated by the hypervariable linker domain.

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.

Rapid functional assays of recombinant IP3 receptors.

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.

Plasma membrane microdomains: organization, function and trafficking.

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.

Dimers of D-myo-inositol 1,4,5-trisphosphate: design, synthesis, and interaction with Ins(1,4,5)P3 receptors.

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.

Synthesis and Ca2+-mobilizing activity of purine-modified mimics of adenophostin A: a model for the adenophostin-Ins(1,4,5)P3 receptor interaction.

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.

Modulation of IP(3)-sensitive Ca(2+) release by 2,3-butanedione monoxime.

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.