Dopamine receptor-interacting protein 78 acts as a molecular chaperone for G gamma subunits before assembly with G beta

Heterotrimeric G proteins play a central role in intracellular communication mediated by extracellular signals, and both G alpha and G beta gamma subunits regulate effectors downstream of activated receptors. The particular constituents of the G protein heterotrimer affect both specificity and efficiency of signal transduction. However, little is known about mechanistic aspects of G protein assembly in the cell that would certainly contribute to formation of heterotrimers of specific composition. It was recently shown that phosducin-like protein (PhLP) modulated both G beta gamma expression and subsequent signaling by chaperoning nascent G beta and facilitating heterodimer formation with G gamma subunits Lukov, G. L., Hu, T., McLaughlin, J. N., Hamm, H. E., and Willardson, B. M. ( 2005) EMBOJ. 24, 1965 - 1975; Humrich, J., Bermel, C., Bunemann, M., Harmark, L., Frost, R., Quitterer, U., and Lohse, M. J. ( 2005) J. Biol. Chem. 280, 20042 - 20050). Here we demonstrate using a variety of techniques that DRiP78, an endoplasmic reticulum resident protein known to regulate the trafficking of several seven transmembrane receptors, interacts specifically with the G gamma subunit but not G beta or G alpha subunits. Furthermore, we demonstrate that DRiP78 and the G beta subunit can compete for the G gamma subunit. DRiP78 also protects G gamma from degradation until a stable partner such as G beta is provided. Furthermore, DRiP78 interaction may represent a mechanism for assembly of specific G beta gamma heterodimers, as selectivity was observed among G gamma isoforms for interaction with DRiP78 depending on the presence of particular G beta subunits. Interestingly, we could detect an interaction between DRiP78 and PhLP, suggesting a role of DRiP78 in the assembly of G beta gamma by linking G gamma to PhLP center dot G beta complexes. Our results, therefore, suggest a role of DRiP78 as a chaperone in the assembly of G beta gamma subunits of the G protein

Seven transmembrane receptor core signaling complexes are assembled prior to plasma membrane trafficking

Much is known about beta(2)-adrenergic receptor trafficking and internalization following prolonged agonist stimulation. However, less is known about outward trafficking of the beta(2)-adrenergic receptor to the plasma membrane or the role that trafficking might play in the assembly of receptor signaling complexes, important for targeting, specificity, and rapidity of subsequent signaling events. Here, by using a combination of bioluminescence resonance energy transfer, bimolecular fluorescence complementation, and confocal microscopy, we evaluated the steps in the formation of the core receptor-G protein heterotrimer complex. By using dominant negative Rab and Sar GTPase constructs, we demonstrate that receptor dimers and receptor-G beta gamma complexes initially associate in the endoplasmic reticulum, whereas G alpha subunits are added to the complex during endoplasmic reticulum-Golgi transit. We also observed that G protein heterotrimers adopt different trafficking itineraries when expressed alone or with stoichiometric co-expression with receptor. Furthermore, deliberate mistargeting of specific components of these complexes leads to diversion of other members from their normal subcellular localization, confirming the role of these early interactions in targeting and formation of specific signaling complexes

Time-Dependent Beneficial Effect of Chronic Polyphenol Treatment with Catechin on Endothelial Dysfunction in Aging Mice

A controlled redox environment is essential for vascular cell maturation and function. During aging, an imbalance occurs, leading to endothelial dysfunction. We hypothesized that, according to the concept of hormesis, exposure to physiologic oxidative stress during the maturation phase of the endothelium will activate protective pathways involved in stress resistance. C57Bl/6 mice were treated with the polyphenol catechin for the last 3 (post-maturation) or 9 months prior study at 12 months of age. Endothelial dysfunction, assessed by acetylcholine-induced dilations of isolated renal arteries, was present at 12 months (P<0.05). Only the 3-month treatment with catechin fully prevented the decline in efficacy and sensitivity to acetylcholine (P<0.05). Splenocytes adhesion to the native endothelium, expression of CD18 and shedding of CD62L and PSGL-1 augmented in 12 months old mice (P<0.05): only 3-month catechin fully normalized adhesion and prevented the expression of adhesion molecules on splenocytes (P<0.05). Aging was associated with vascular gene alterations, which were prevented by 3-month catechin treatment (P<0.05). In contrast, 9-month catechin further increased COX-2, p22phox and reduced MnSOD (P<0.05). In conclusion, we demonstrate a pivotal role of cellular redox equilibrium: exposure to physiologic oxidative stress during the maturation phase of the endothelium is essential for its function

Photoreleasable ligands to study intracrine angiotensin II signalling.

International audienceThe renin-angiotensin system plays a key role in cardiovascular physiology and its overactivation has been implicated in the pathogenesis of several major cardiovascular diseases. There is growing evidence that angiotensin II (Ang-II) may function as an intracellular peptide to activate intracellular/nuclear receptors and their downstream signalling effectors independently of cell surface receptors. Current methods used to study intracrine Ang-II signalling are limited to indirect approaches because of a lack of selective intracellularly-acting probes. Here, we present novel photoreleasable Ang-II analogues used to probe intracellular actions with spatial and temporal precision. The photorelease of intracellular Ang-II causes nuclear and cytosolic calcium mobilization and initiates the de novo synthesis of RNA in cardiac cells, demonstrating the application of the method. Several lines of evidence suggest that intracellular angiotensin II (Ang-II) contributes to the regulation of cardiac contractility, renal salt reabsorption, vascular tone and metabolism; however, work on intracrine Ang-II signalling has been limited to indirect approaches because of a lack of selective intracellularly-acting probes. Here, we aimed to synthesize and characterize cell-permeant Ang-II analogues that are inactive without uncaging, but release active Ang-II upon exposure to a flash of UV-light, and act as novel tools for use in the study of intracrine Ang-II physiology. We prepared three novel caged Ang-II analogues, [Tyr(DMNB)(4)]Ang-II, Ang-II-ODMNB and [Tyr(DMNB)(4)]Ang-II-ODMNB, based upon the incorporation of the photolabile moiety 4,5-dimethoxy-2-nitrobenzyl (DMNB). Compared to Ang-II, the caged Ang-II analogues showed 2-3 orders of magnitude reduced affinity toward both angiotensin type-1 (AT1R) and type-2 (AT2R) receptors in competition binding assays, and greatly-reduced potency in contraction assays of rat thoracic aorta. After receiving UV-irradiation, all three caged Ang-II analogues released Ang-II and potently induced the contraction of rat thoracic aorta. [Tyr(DMNB)(4)]Ang-II showed the most rapid photolysis upon UV-irradiation and was the focus of subsequent characterization. Whereas Ang-II and photolysed [Tyr(DMNB)(4)]Ang-II increased ERK1/2 phosphorylation (via AT1R) and cGMP production (AT2R), caged [Tyr(DMNB)(4)]Ang-II did not. Cellular uptake of [Tyr(DMNB)(4)]Ang-II was 4-fold greater than that of Ang-II and significantly greater than uptake driven by the positive-control HIV TAT(48-60) peptide. Intracellular photolysis of [Tyr(DMNB)(4)]Ang-II induced an increase in nucleoplasmic Ca(2+) ([Ca(2+)]n), and initiated 18S rRNA and nuclear factor kappa B mRNA synthesis in adult cardiac cells. We conclude that caged Ang-II analogues represent powerful new tools for use in the selective study of intracrine signalling via Ang-II