The enzyme phospholipase A2 (cPLA2α) is involved in the formation of intercisternal tubules that mediate transport of proteins within the Golgi complex.

In this study, we investigated the regulation of different G protein-coupled receptor (GPCR)-stimulated signaling pathways by GPCR kinase 2 (GRK2). We used thyrotropin receptor, which is coupled to different G proteins, to investigate the regulation of G␣s-and G␣q-mediated signaling (assessed by cAMP and inositol phosphate production, respectively). In transfected cells, both pathways were desensitized by GRK2. However a kinase-dead GRK2 mutant (GRK2-K220R) only decreased inositol phosphate production, indicating that GRK2 could regulate G␣q signaling through a phosphorylation-independent mechanism. Similar results were obtained with serotonin receptor 5-hydroxytryptamine 2C , which is coupled to G␣q. This effect was mimicked by the N-terminal domain of GRK2 (GRK2-Nter), but not by the C-terminal domain. In cells transfected with G␣q, direct activation of G␣q signaling (by AlF 4 Ϫ ) was desensitized by GRK2-Nter, indicating an effect at the G␣-level. For comparison, in parallel samples we studied a protein regulator of G protein signaling RGS4 and we found a similar regulatory profile. We therefore hypothesized that the GRK2-Nter could directly interact with the G␣q subunit to regulate its signaling, as demonstrated for several RGS proteins. This hypothesis is further supported by the presence, within the GRK2-Nter, of an RGS homology domain. In direct binding experiments, we found that GRK2-Nter interacts with G␣q (only when activated) but not with G␣s and G␣o. We conclude that GRK2, besides desensitizing the GPCR by phosphorylation, is able to selectively bind to G␣q and to regulate its signaling.

Large pleiomorphic carriers leave the Golgi complex for the plasma membrane by en bloc extrusion of specialized tubular domains, which then undergo fission. Several components of the underlying molecular machinery have been identified, including those involved in the budding/initiation of tubular carrier precursors (for example, the phosphoinositide kinase PI(4)KIIIβ, the GTPase ARF, and FAPP2), and in the fission of these precursors (for example, PKD, CtBP1-S/BARS). However, how these proteins interact to bring about carrier formation is poorly understood. Here, we describe a protein complex that mediates carrier formation and contains budding and fission molecules, as well as other molecules, such as the adaptor protein 14-3-3γ. Specifically, we show that 14-3-3γ dimers bridge CtBP1-S/BARS with PI(4)KIIIβ, and that the resulting complex is stabilized by phosphorylation by PKD and PAK. Disrupting the association of these proteins inhibits the fission of elongating carrier precursors, indicating that this complex couples the carrier budding and fission processes.

The glycerophosphodiester phosphodiesterases are evolutionarily conserved proteins that have been linked to several patho/physiological functions, comprising bacterial pathogenicity and mammalian cell proliferation or differentiation. The bacterial enzymes do not show preferential substrate selectivities among the glycerophosphodiesters, and they are mainly dedicated to glycerophosphodiester hydrolysis, producing glycerophosphate and alcohols as the building blocks that are required for bacterial biosynthetic pathways. In some cases, this enzymatic activity has been demonstrated to contribute to bacterial pathogenicity, such as with Hemophilus influenzae. Mammalian glyerophosphodiesterases have high substrate specificities, even if the number of potential physiological substrates is continuously increasing. Some of these mammalian enzymes have been directly linked to cell differentiation, such as GDE2, which triggers motor neuron differentiation, and GDE3, the enzymatic activity of which is necessary and sufficient to induce osteoblast differentiation. Instead, GDE5 has been shown to inhibit skeletal muscle development independent of its enzymatic activity.

The metabotropic glutamate 1 (mGlu 1 ) receptor in cerebellar Purkinje cells plays a key role in motor learning and motor coordination. Here we show that the G proteincoupled receptor kinases (GRK) 2 and 4, which are expressed in these cells, regulate the mGlu 1 receptor by at least in part different mechanisms. Using kinase-dead mutants in HEK293 cells, we found that GRK4, but not GRK2, needs the intact kinase activity to desensitize the mGlu 1 receptor, whereas GRK2, but not GRK4, can interact with and regulate directly the activated G␣ q . In cells transfected with GRK4 and exposed to agonist, ␤-arrestin was first recruited to plasma membranes, where it was co-localized with the mGlu 1 receptor, and then internalized in vesicles. The receptor was also internalized but in different vesicles. The expression of ␤-arrestin V53D dominant negative mutant, which did not affect the mGlu 1 receptor internalization, reduced by 70 -80% the stimulation of mitogen-activated protein (MAP) kinase activation by the mGlu 1 receptor. The agonist-stimulated differential sorting of the mGlu 1 receptor and ␤-arrestin as well as the activation of MAP kinases by mGlu 1 agonist was confirmed in cultured cerebellar Purkinje cells. A major involvement of GRK4 and of ␤-arrestin in agonist-dependent receptor internalization and MAP kinase activation, respectively, was documented in cerebellar Purkinje cells using an antisense treatment to knock down GRK4 and expressing ␤-arrestin V53D dominant negative mutant by an adenovirus vector. We conclude that GRK2 and GRK4 regulate the mGlu 1 receptor by different mechanisms and that ␤-arrestin is directly involved in glutamate-stimulated MAP kinase activation by acting as a signaling molecule.Metabotropic glutamate (mGlu) 1 receptors, which are activated by the excitatory amino acid glutamate, are part of an original family of G protein-coupled receptors (GPCR) called the family 3 GPCRs (1-3). These include all the mGlu receptor subtypes, Ca 2ϩ -sensing and GABA B receptors, and some putative olfactory, pheromone, and taste receptors. Eight subtypes of mGlu receptors have been identified, which are implicated in different aspects of physiology and pathology of the central nervous system. Group I mGlu receptors (mGlu 1 and mGlu 5 ), which stimulate polyphosphoinositide hydrolysis by coupling to G q , are localized in the peripheral parts of postsynaptic dendrites and contribute to the regulation of synaptic plasticity. For example, mGlu 1 receptor present in cerebellar Purkinje cells plays a key role in motor learning and motor coordination. Similar to many other GPCRs, the signal transduction of the mGlu 1 receptor is strictly regulated by multiple mechanisms acting at different levels of signal propagation (1). After prolonged or repeated stimulation, receptors are profoundly desensitized. Protein kinase C is clearly involved in this process, although a protein kinase C-independent component of mGlu 1 receptor desensitization was also observed (4). The activated ␣ subunit of the G q (G␣ q ) ca...

Invadopodia are proteolytically active membrane protrusions that extend from the ventral surface of invasive tumoral cells grown on an extracellular matrix (ECM). The core machinery controlling invadopodia biogenesis is regulated by the Rho GTPase Cdc42. To understand the upstream events regulating invadopodia biogenesis, we investigated the role of Fgd1, a Cdc42-specific guanine nucleotide exchange factor. Loss of Fgd1 causes the rare inherited human developmental disease faciogenital dysplasia. Here, we show that Fgd1 is required for invadopodia biogenesis and ECM degradation in an invasive cell model and functions by modulation of Cdc42 activation. We also find that Fgd1 is expressed in human prostate and breast cancer as opposed to normal tissue and that expression levels matched tumor aggressiveness. Our findings suggest a central role for Fgd1 in the focal degradation of the ECM in vitro and, for the first time, show a connection between Fgd1 and cancer progression, proposing that it might function during tumorigenesis. [Cancer Res 2009;69(3):747-52]

Mammalian glycerophosphodiester phosphodiesterases (GPPDEs) have been identified recently and shown to be implicated in several physiological functions. This study isolated a novel GP-PDE, GDE5, and showed that GDE5 selectively hydrolyzes glycerophosphocholine (GroPCho) and controls skeletal muscle development. We show that GDE5 expression was reduced in atrophied skeletal muscles in mice and that decreasing GDE5 abundance promoted myoblastic differentiation, suggesting that decreased GDE5 expression has a counter-regulatory effect on the progression of skeletal muscle atrophy. Forced expression of full-length GDE5 in cultured myoblasts suppressed myogenic differentiation. Unexpectedly, a truncated GDE5 construct (GDE5⌬C471), which contained a GP-PDE sequence identified in other GP-PDEs but lacked GroPCho phosphodiesterase activity, showed a similar inhibitory effect. Furthermore, transgenic mice specifically expressing GDE5⌬C471 in skeletal muscle showed less skeletal muscle mass, especially type II fiber-rich muscle. These results indicate that GDE5 negatively regulates skeletal muscle development even without GroPCho phosphodiesterase activity, providing novel insight into the biological significance of mammalian GP-PDE function in a non-enzymatic mechanism.

The phosphoinositides have well-defined roles in the control of cellular functions, including cytoskeleton dynamics, membrane trafficking, and cell signaling. However, the interplay among the phosphoinositides and their diffusible derivatives that originate through phospholipase A2 action (the lysophosphoinositides and glycerophosphoinositols) remains to be fully elucidated. Here we demonstrate that in PCCl3 rat thyroid cells, the intracellular levels of glycerophosphoinositol are finely modulated by ATP and norepinephrine through the P2Y metabotropic and alpha-adrenergic receptors, respectively. The enzyme involved here is phospholipase A2 IValpha (PLA2 IValpha), which in these cells specifically hydrolyzes phosphatidylinositol, forming lysophosphatidylinositol, glycerophosphoinositol, and arachidonic acid. This receptor-mediated activation of PLA2 IValpha leads to stimulation of PCCl3 cell growth. The involvement of a PLA2 IValpha-mediated pathway is demonstrated by inhibition of the increase in intracellular glycerophosphoinositol levels and cell proliferation by specific inhibitors, RNA interference, and overexpression of the dominant-negative PLA2 IValpha(1-522). Modulation of PCCl3 cell growth is not seen with inhibitors of arachidonic acid metabolism. In conclusion, these data characterize glycerophosphoinositol as a mediator of the purinergic and adrenergic regulation of PCCl3 cell proliferation, defining a novel regulatory cascade specifically involving this soluble phosphoinositide derivative and widening the involvement of the phosphoinositides in the regulation of cell function.