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Metastasis is a significant health issue. The standard mode of care is combination of chemotherapy and targeted therapeutics but the 5-year survival rate remains low. New/better drug targets that can improve outcomes of patients with metastatic disease are needed. Metastasis is a complex process, with each step conferred by a set of genetic aberrations. Mapping the molecular changes associated with metastasis improves our understanding of the etiology of this disease and contributes to the pipeline of targeted therapeutics. Here, phosphoproteomics of a xenograft-derived in vitro model comprising 4 isogenic cell lines with increasing metastatic potential implicated Transient Receptor Potential Vanilloid subtype 4 in breast cancer metastasis. TRPV4 mRNA levels in breast, gastric and ovarian cancers correlated with poor clinical outcomes, suggesting a wide role of TRPV4 in human epithelial cancers. TRPV4 was shown to be required for breast cancer cell invasion and transendothelial migration but not growth/proliferation. Knockdown of Trpv4 significantly reduced the number of metastatic nodules in mouse xenografts leaving the size unaffected. Overexpression of TRPV4 promoted breast cancer cell softness, blebbing, and actin reorganization. The findings provide new insights into the role of TRPV4 in cancer extravasation putatively by reducing cell rigidity through controlling the cytoskeleton at the cell cortex.

1 TRPM2 is a Ca2+‐permeable nonselective cation channel activated by intracellular ADP‐ribose (ADPR) and by hydrogen peroxide (H2O2). We investigated the modulation of TRPM2 activity by N‐(p‐amylcinnamoyl)anthranilic acid (ACA). ACA has previously been reported to inhibit phospholipase A2 (PLA2). 2 Using patch‐clamp and calcium‐imaging techniques, we show that extracellular application of 20 μM ACA completely blocked ADPR‐induced whole‐cell currents and H2O2‐induced Ca2+ signals (IC50=1.7 μM) in HEK293 cells transfected with human TRPM2. Two other PLA2 inhibitors, p‐bromophenacyl bromide (BPB; 100 μM) and arachidonyl trifluoromethyl ketone (20 μM), had no significant effect on ADPR‐stimulated TRPM2 activity. 3 Inhibition of TRPM2 whole‐cell currents by ACA was voltage independent and accelerated at decreased pH. ACA was ineffective when applied intracellularly. The single‐channel conductance was not changed during ACA treatment, suggesting a reduction of TRPM2 open probability by modulating channel gating. 4 ACA (20 μM) also blocked currents through human TRPM8 and TRPC6 expressed in HEK293 cells, while BPB (100 μM) was ineffective. TRPC6‐mediated currents (IC50=2.3 μM) and TRPM8‐induced Ca2+ signals (IC50=3.9 μM) were blocked in a concentration‐dependent manner. 5 ADPR‐induced currents in human U937 cells, endogeneously expressing TRPM2 protein, were fully suppressed by 20 μM ACA. 6 Our data indicate that ACA modulates the activity of different TRP channels independent of PLA2 inhibition. Owing to its high potency and efficacy ACA can serve, in combination with other blockers, as a useful tool for studying the unknown function of TRPM2 in native cells. British Journal of Pharmacology (2006) 148, 264–273. doi:10.1038/sj.bjp.0706739

The mammalian melastatin-related transient receptor potential (TRPM) subfamily contains eight members. TRPM proteins, consisting of six putative transmembrane domains and intracellular N and C termini, form monovalent-permeable cation channels with variable selectivity for Ca(2+), Mg(2+) and other divalent cations. Some functions are linked to their individual cation selectivity: the highly divalent-permeable cation channels TRPM6 and TRPM7 are involved in the control of Mg(2+) influx, whereas the Ca(2+)-impermeable channels TRPM4 and TRPM5 modulate cellular Ca(2+) entry by determining the membrane potential. TRPM2, TRPM3 and TRPM8 mediate a direct influx of Ca(2+) in response to specific stimuli. Electrophysiological properties of the founding member, melastatin (TRPM1), are unexplored. The individual TRPM members are activated by different stimuli, including voltage, Ca(2+), temperature, cell swelling, lipid compounds and other endogenous or exogenous ligands. This review summarizes molecular features, activation mechanisms, biophysical properties and modulators of TRPM channels.

Phosphoinositide 3-kinase gamma (PI3Kγ) has profound roles downstream of G-protein–coupled receptors in inflammation, cardiac function, and tumor progression. To gain insight into how the enzyme’s activity is shaped by association with its p101 adaptor subunit, lipid membranes, and Gβγ heterodimers, we mapped these regulatory interactions using hydrogen–deuterium exchange mass spectrometry. We identify residues in both the p110γ and p101 subunits that contribute critical interactions with Gβγ heterodimers, leading to PI3Kγ activation. Mutating Gβγ-interaction sites of either p110γ or p101 ablates G-protein–coupled receptor-mediated signaling to p110γ/p101 in cells and severely affects chemotaxis and cell transformation induced by PI3Kγ overexpression. Hydrogen–deuterium exchange mass spectrometry shows that association with the p101 regulatory subunit causes substantial protection of the RBD-C2 linker as well as the helical domain of p110γ. Lipid interaction massively exposes that same helical site, which is then stabilized by Gβγ. Membrane-elicited conformational change of the helical domain could help prepare the enzyme for Gβγ binding. Our studies and others identify the helical domain of the class I PI3Ks as a hub for diverse regulatory interactions that include the p101, p87 (also known as p84), and p85 adaptor subunits; Rab5 and Gβγ heterodimers; and the β-adrenergic receptor kinase.

In blood vessels, the endothelium is a crucial signal transduction interface in control of vascular tone and blood pressure to ensure energy and oxygen supply according to the organs' needs. In response to vasoactive factors and to shear stress elicited by blood flow, the endothelium secretes vasodilating or vasocontracting autacoids, which adjust the contractile state of the smooth muscle. In endothelial sensing of shear stress, the osmo- and mechanosensitive Ca(2+)-permeable TRPV4 channel has been proposed to be candidate mechanosensor. Using TRPV4(-/-) mice, we now investigated whether the absence of endothelial TRPV4 alters shear-stress-induced arterial vasodilation. In TRPV4(-/-) mice, loss of the TRPV4 protein was confirmed by Western blot, immunohistochemistry and by in situ-patch-clamp techniques in carotid artery endothelial cells (CAEC). Endothelium-dependent vasodilation was determined by pressure myography in carotid arteries (CA) from TRPV4(-/-) mice and wild-type littermates (WT). In WT CAEC, TRPV4 currents could be elicited by TRPV4 activators 4alpha-phorbol-12,13-didecanoate (4alphaPDD), arachidonic acid (AA), and by hypotonic cell swelling (HTS). In striking contrast, in TRPV4(-/-) mice, 4alphaPDD did not produce currents and currents elicited by AA and HTS were significantly reduced. 4alphaPDD caused a robust and endothelium-dependent vasodilation in WT mice, again conspicuously absent in TRPV4(-/-) mice. Shear stress-induced vasodilation could readily be evoked in WT, but was completely eliminated in TRPV4(-/-) mice. In addition, flow/reperfusion-induced vasodilation was significantly reduced in TRPV4(-/-) vs. WT mice. Vasodilation in response to acetylcholine, vasoconstriction in response to phenylephrine, and passive mechanical compliance did not differ between genotypes, greatly underscoring the specificity of the above trpv4-dependent phenotype for physiologically relevant shear stress. Genetically encoded loss-of-function of trpv4 results in a loss of shear stress-induced vasodilation, a response pattern critically dependent on endothelial TRPV4 expression. Thus, Ca(2+)-influx through endothelial TRPV4 channels is a molecular mechanism contributing significantly to endothelial mechanotransduction.

1 2‐Aminoethoxydiphenyl borate (2‐APB) has been widely used to examine the roles of inositol 1,4,5‐trisphosphate receptors (IP3Rs) and store‐operated Ca2+ entry and is an emerging modulator of cationic channels encoded by transient receptor potential (TRP) genes. 2 Using Ca2+‐indicator dye and patch‐clamp recording we first examined the blocking effect of 2‐APB on human TRPC5 channels expressed in HEK‐293 cells. 3 The concentration–response curve has an IC50 of 20 μM and slope close to 1.0, suggesting one 2‐APB molecule binds per channel. The blocking effect is not shared by other Ca2+ channel blockers including methoxyverapamil, nifedipine, N‐propargylnitrendipine, or berberine. 4 In whole‐cell and excised membrane patch recordings, 2‐APB acts from the extracellular but not intracellular face of the membrane. 5 Block of TRPC5 by 2‐APB is less at positive voltages, suggesting that it enters the electric field or acts by modulating channel gating. 6 2‐APB also blocks TRPC6 and TRPM3 expressed in HEK‐293 cells, but not TRPM2. 7 Block of TRP channels by 2‐APB may be relevant to cell proliferation because 2‐APB has a greater inhibitory effect on proliferation in cells overexpressing TRPC5. 8 Our data indicate a specific and functionally important binding site on TRPC5 that enables block by 2‐APB. The site is only available via an extracellular route and the block shows mild voltage‐dependence. British Journal of Pharmacology (2005) 145, 405–414. doi:10.1038/sj.bjp.0706197

The prohibitin (PHB)-domain proteins are membrane proteins that regulate a variety of biological activities, including mechanosensation, osmotic homeostasis, and cell signaling, although the mechanism of this regulation is unknown. We have studied two members of this large protein family, MEC-2, which is needed for touch sensitivity in Caenorhabditis elegans, and Podocin, a protein involved in the function of the filtration barrier in the mammalian kidney, and find that both proteins bind cholesterol. This binding requires the PHB domain (including palmitoylation sites within it) and part of the N-terminally adjacent hydrophobic domain that attaches the proteins to the inner leaflet of the plasma membrane. By binding to MEC-2 and Podocin, cholesterol associates with ion-channel complexes to which these proteins bind: DEG/ENaC channels for MEC-2 and TRPC channels for Podocin. Both the MEC-2-dependent activation of mechanosensation and the Podocin-dependent activation of TRPC channels require cholesterol. Thus, MEC-2, Podocin, and probably many other PHB-domain proteins by binding to themselves, cholesterol, and target proteins regulate the formation and function of large protein-cholesterol supercomplexes in the plasma membrane.

Eukaryotic cells respond to many hormones and neurotransmitters with increased activity of the enzyme phospholipase C and a subsequent rise in the concentration of intracellular free calcium ([Ca 2+ ] i ) 1 . The increase in [Ca 2+ ] i occurs as a result of the release of Ca 2+ from intracellular stores and an influx of Ca 2+ through the plasma membrane 2 , 3 , 4 ; this influx of Ca 2+ may 5 or may not 6 be store-dependent. Drosophila transient receptor potential (TRP) proteins and some mammalian homologues (TRPC proteins) are thought to mediate capacitative Ca 2+ entry 7 , 8 , 9 . Here we describe the molecular mechanism of store-depletion-independent activation of a subfamily of mammalian TRPC channels. We find that hTRPC6 is a non-selective cation channel that is activated by diacylglycerol in a membrane-delimited fashion, independently of protein kinases C activated by diacylglycerol. Although hTRPC3, the closest structural relative of hTRPC6, is activated in the same way, TRPCs 1, 4 and 5 and the vanilloid receptor subtype 1 are unresponsive to the lipid mediator. Thus, hTRPC3 and hTRPC6 represent the first members of a new functional family of second-messenger-operated cation channels, which are activated by diacylglycerol.

Psoriasis is a characteristic inflammatory and scaly skin condition with typical histopathological features including increased proliferation and hampered differentiation of keratinocytes. The activation of innate and adaptive inflammatory cellular immune responses is considered to be the main trigger factor of the epidermal changes in psoriatic skin. However, the molecular players that are involved in enhanced proliferation and impaired differentiation of psoriatic keratinocytes are only partly understood. One important factor that regulates differentiation on the cellular level is Ca(2+). In normal epidermis, a Ca(2+) gradient exists that is disturbed in psoriatic plaques, favoring impaired keratinocyte proliferation. Several TRPC channels such as TRPC1, TRPC4, or TRPC6 are key proteins in the regulation of high [Ca(2+)](ex) induced differentiation. Here, we investigated if TRPC channel function is impaired in psoriasis using calcium imaging, RT-PCR, western blot analysis and immunohistochemical staining of skin biopsies. We demonstrated substantial defects in Ca(2+) influx in psoriatic keratinocytes in response to high extracellular Ca(2+) levels, associated with a downregulation of all TRPC channels investigated, including TRPC6 channels. As TRPC6 channel activation can partially overcome this Ca(2+) entry defect, specific TRPC channel activators may be potential new drug candidates for the topical treatment of psoriasis.

Class IB phosphoinositide 3-kinase γ (PI3Kγ) elicits various immunologic and cardiovascular responses; however, the molecular basis for this signal heterogeneity is unclear. PI3Kγ consists of a catalytic p110γ and a regulatory p87PIKAP (p87, also p84) or p101 subunit. Hitherto p87 and p101 are generally assumed to exhibit redundant functions in receptor-induced and G protein βγ (Gβγ)-mediated PI3Kγ regulation. Here we investigated the molecular mechanism for receptor-dependent p87/p110γ activation. By analyzing GFP-tagged proteins expressed in HEK293 cells, PI3Kγ-complemented bone marrow-derived mast cells (BMMCs) from p110γ⁻/⁻ mice, and purified recombinant proteins reconstituted to lipid vesicles, we elucidated a novel pathway of p87-dependent, G protein-coupled receptor (GPCR)-induced PI3Kγ activation. Although p101 strongly interacted with Gβγ, thereby mediating PI3Kγ membrane recruitment and stimulation, p87 exhibited only a weak interaction, resulting in modest kinase activation and lack of membrane recruitment. Surprisingly, Ras-GTP substituted the missing Gβγ-dependent membrane recruitment of p87/p110γ by direct interaction with p110γ, suggesting the indispensability of Ras for activation of p87/p110γ. Consequently, interference with Ras signaling indeed selectively blocked p87/p110γ, but not p101/p110γ, kinase activity in HEK293 and BMMC cells, revealing an important crosstalk between monomeric and trimeric G proteins for p87/p110γ activation. Our data display distinct signaling requirements of p87 and p101, conferring signaling specificity to PI3Kγ that could open up new possibilities for therapeutic intervention.