A novel GRK2/HDAC6 interaction modulates cell spreading and motility.

Cell motility and adhesion involves dynamic microtubule (MT) acetylation/deacetylation, a process regulated by enzymes as HDAC6, a major cytoplasmic α-tubulin deacetylase. We identify G protein-coupled receptor kinase 2 (GRK2) as a key novel stimulator of HDAC6. GRK2, which levels inversely correlate with the extent of α-tubulin acetylation in epithelial cells and fibroblasts, directly associates with and phosphorylates HDAC6 to stimulate α-tubulin deacetylase activity. Remarkably, phosphorylation of GRK2 itself at S670 specifically potentiates its ability to regulate HDAC6. GRK2 and HDAC6 colocalize in the lamellipodia of migrating cells, leading to local tubulin deacetylation and enhanced motility. Consistently, cells expressing GRK2-K220R or GRK2-S670A mutants, unable to phosphorylate HDAC6, exhibit highly acetylated cortical MTs and display impaired migration and protrusive activity. Finally, we find that a balanced, GRK2/HDAC6-mediated regulation of tubulin acetylation differentially modulates the early and late stages of cellular spreading. This novel GRK2/HDAC6 functional interaction may have important implications in pathological contexts.

G protein-coupled receptor kinase 2 positively regulates epithelial cell migration.

Cell migration requires integration of signals arising from both the extracellular matrix and messengers acting through G protein-coupled receptors (GPCRs). We find that increased levels of G protein-coupled receptor kinase 2 (GRK2), a key player in GPCR regulation, potentiate migration of epithelial cells towards fibronectin, whereas such process is decreased in embryonic fibroblasts from hemizygous GRK2 mice or upon knockdown of GRK2 expression. Interestingly, the GRK2 effect on fibronectin-mediated cell migration involves the paracrine/autocrine activation of a sphingosine-1-phosphate (S1P) Gi-coupled GPCR. GRK2 positively modulates the activity of the Rac/PAK/MEK/ERK pathway in response to adhesion and S1P by a mechanism involving the phosphorylation-dependent, dynamic interaction of GRK2 with GIT1, a key scaffolding protein in cell migration processes. Furthermore, decreased GRK2 levels in hemizygous mice result in delayed wound healing rate in vivo, consistent with a physiological role of GRK2 as a regulator of coordinated integrin and GPCR-directed epithelial cell migration.

The G protein-coupled receptor kinase (GRK) interactome: role of GRKs in GPCR regulation and signaling.

G protein-coupled receptor kinases (GRKs) and arrestins are key participants in the canonical pathways leading to phosphorylation-dependent GPCR desensitization, endocytosis, intracellular trafficking and resensitization as well as in the modulation of important intracellular signaling cascades by GPCR. Novel studies have revealed a phosphorylation-independent desensitization mechanism operating through their RGS-homology (RH) domain and the recent determination of the crystal structures of GRK2 and GRK6 has uncovered interesting details on the structure-function relationships of these kinases. Emerging evidence indicates that the activity of GRKs is tightly modulated by mechanisms including phosphorylation by different kinases and interaction with several cellular proteins such as calmodulin, caveolin or RKIP. In addition, GRKs are involved in multiple interactions with non-receptor proteins (PI3K, Akt, GIT or MEK) that point to novel GRK cellular roles. In this article, our purpose is to describe the ever increasing map of functional interactions for GRK proteins as a basis to better understand its contribution to cellular processes.

G protein-coupled receptor kinase 2 (GRK2) in migration and inflammation.

G protein-coupled receptor kinase 2 (GRK2) is a key modulator of G protein-coupled receptors and other plasma membrane receptors stimulated by chemotactic messengers. On top of that, GRK2 has been reported to interact with a variety of signal transduction proteins related to cell migration such as MEK, Akt, PI3Kgamma or GIT. Interestingly, the levels of expression and activity of this kinase are altered in a number of inflammatory disorders (as rheumatoid arthritis or multiple sclerosis), thus suggesting that it may play an important role in the onset or development of these pathologies. This review summarizes the mechanisms involved in the control of GRK2 expression and function and highlights novel functional interactions of this protein that might help to explain how altered GRK2 levels affects cell migration in different cell types and pathological settings.

Altered expression of nucleoside transporter genes (SLC28 and SLC29) in adipose tissue from HIV-1-infected patients.

BACKGROUND: Nucleoside transporter proteins (NTs) encoded by members of the SLC28 and SLC29 gene families contribute to nucleoside and nucleobase recycling but also modulate extracellular adenosine levels and thus adenosine-regulated metabolic targets. METHODS: We have examined the expression pattern of NT-encoding genes in human adipose tissue and we have further analysed whether the mRNA related to these genes show changes in their amounts associated with either HIV-1 infection, highly active antiretroviral therapy (HAART) or development of HIV-1-associated lipodystrophy syndrome (HALS). RESULTS: Human adipocytes express SLC28A1, SLC28A2 and SLC28A3 (encoding hCNT1, hCNT2 and hCNT3, respectively) and SLC29A1 and SLC29A2 (encoding hENT1 and hENT2, respectively). HIV-1 infection, prior to HAART and HALS development, is associated with the upregulation of the mRNA levels of the genes encoding hCNT1, hCNT3 and hENT2. The increase in the mRNA amounts for the former two genes may be due to the action of tumour necrosis factor-alpha (TNF-alpha), a cytokine with enhanced expression in adipose tissue following HIV-1 infection, as the effect is also observed in human adipocytes in culture after treatment with TNF-alpha. HAART and HALS development are associated with the upregulation of the mRNA levels encoding hCNT2 and hENT1, and further enhancement of hCNT1, hCNT3 and hENT2 gene expression. CONCLUSIONS: These data suggest that selected genes of the SLC28 and SLC29 families are not only targets of HIV-1 infection, but might also contribute to the development of adipose tissue alterations leading to lipodystrophy.

Bile acids alter the subcellular localization of CNT2 (concentrative nucleoside cotransporter) and increase CNT2-related transport activity in liver parenchymal cells.

CNT2 (concentrative nucleoside cotransporter) is a plasma membrane high-affinity Na+-coupled adenosine transporter, also localized in intracellular structures. This transporter protein may play additional roles other than nucleoside salvage, since it has recently been shown to be under purinergic control via K(ATP) channels, by a mechanism that does not seem to involve changes in its subcellular localization. In an attempt to identify the agents that promote CNT2 trafficking, bile acids were found to increase CNT2-related transport activity in a K(ATP) channel-independent manner in both Fao hepatoma and rat liver parenchymal cells. A maximum effect was recorded after treatment with hydrophilic anions such as TCA (taurocholate). However, this effect did not involve changes in the amount of CNT2 protein, it was instead associated with a subcellular redistribution of CNT2, resulting in an accumulation of the transporter at the plasma membrane. This was deduced from subcellular fractionation studies, biotinylation of plasma membrane proteins and subsequent CNT2 detection in streptavidin precipitates and in vivo confocal microscopic analysis of the distribution of a YFP (yellow fluorescent protein)-CNT2 construct. The induction of CNT2 translocation, triggered by TCA, was inhibited by wortmannin, dibutyryl-AMPc, PD98059 and colchicine, thus suggesting the involvement of the PI3K/ERK (phosphoinositide 3-kinase/extracellular-signal related kinase) pathway in microtubule-dependent activation of recombinant CNT2. These are novel effects of bile-acid physiology and provide the first evidence for short-term regulation of CNT2 translocation into and from the plasma membrane.

Long term endocrine regulation of nucleoside transporters in rat intestinal epithelial cells.

We studied the regulation of nucleoside transporters in intestinal epithelial cells upon exposure to either differentiating or proliferative agents. Rat intestinal epithelial cells (line IEC-6) were incubated in the presence of differentiating (glucocorticoids) or proliferative (EGF and TGF-alpha) agents. Nucleoside uptake rates and nucleoside transporter protein and mRNA levels were assessed. The signal transduction pathways used by the proliferative stimuli were analyzed. We found that glucocorticoids induce an increase in sodium-dependent, concentrative nucleoside transport rates and in protein and mRNA levels of both rCNT2 and rCNT1, with negligible effects on the equilibrative transporters. EGF and TGF-alpha induce an increase in the equilibrative transport rate, mostly accounted for by an increase in rENT1 activity and mRNA levels, rENT2 mRNA levels remaining unaltered. This effect is mimicked by another proliferative stimulus that functions as an in vitro model of epithelial wounding. Here, rENT1 activity and mRNA levels are also increased, although the signal transduction pathways used by the two stimuli are different. We concluded that differentiation of rat intestinal epithelial cells is accompanied by increased mature enterocyte features, such as concentrative nucleoside transport (located at the brush border membrane of the enterocyte), thus preparing the cell for its ultimate absorptive function. A proliferative stimulus induces the equilibrative nucleoside activities (mostly through ENT1) known to be located at the basolateral membrane, allowing the uptake of nucleosides from the bloodstream for the increased demands of the proliferating cell.

New roles of G protein-coupled receptor kinase 2 (GRK2) in cell migration.

G protein-coupled receptor kinase 2 (GRK2) was initially identified as a key player, together with beta-arrestins, in the regulation of multiple G protein-coupled receptors (GPCR). Further research has revealed a complex GRK2 interactome, that includes a variety of proteins related to cell motility, and a role for GRK2 kinase activity in inhibiting chemokine-induced immune cell migration. In addition, we have recently reported that GRK2 positively regulates integrin and sphingosine-1-phosphate-dependent motility in epithelial cell types and fibroblasts, acting as a scaffold molecule. We suggest that the positive or negative correlation of GRK2 levels with cell migration would depend on the cell type, specific stimuli acting through plasma membrane receptors, or on the signalling context, leading to differential networks of interaction of GRK2 with cell migration-related signalosomes.

Extracellular adenosine activates AMP-dependent protein kinase (AMPK).

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a heterotrimeric complex that senses intracellular energy status and exerts rapid regulation on energy-demanding and -consuming metabolic pathways. Although alterations in the intracellular adenosine nucleotide pool are traditionally assumed to be the consequence of changes in energy metabolism, in this study we have addressed the question of whether extracellular adenosine contributes to AMPK regulation. In the intestinal rat epithelial cell line IEC-6, addition of adenosine rapidly increases AMP intracellular concentrations and upregulates alpha1AMPK, thus promoting phosphorylation of its downstream target acetyl-CoA carboxylase (ACC). The effect of adenosine on AMPK signaling is completely blocked by transducing IEC-6 cells with an adenoviral vector expressing a mutated alpha1 subunit, resulting in a dominant-negative effect on endogenous AMPK activity. These effects are blocked by 5'-iodotubercidine (5'-ITU), an inhibitor of adenosine kinase. Moreover, inhibition of adenosine transport through the concentrative adenosine plasma membrane transporter CNT2 with formycin B results in the blockade of adenosine-mediated AMPK signaling. Extracellular adenosine is equally able to activate AMPK and promote ACC phosphorylation in liver parenchymal cell models in a manner that is also inhibited by 5'-ITU. In summary, this study shows that adenosine, when added at physiological concentrations, activates AMPK and promotes ACC phosphorylation. Adenosine must be transported and phosphorylated to exert its action. Thus, nucleoside transporters might be novel players in the complex regulation of AMPK and energy metabolism.