The Toll-like receptor 2/1 (TLR2/1) complex initiates human platelet activation via the src/Syk/LAT/PLCγ2 signalling cascade

The specific TLR2/1 complex activator Pam3CSK4 has been shown to provoke prominent activation and aggregation of human non-nucleated platelets. As Pam3CSK4-evoked platelet activation does not employ the major signalling pathway established in nucleated immune cells, we investigated if the TLR2/1 complex on platelets may initiate signalling pathways known to be induced by physiological agonists such as collagen via GPVI or thrombin via PARs. We found that triggering TLR2/1 complex-signalling with Pam3CSK4, in common with that induced via GPVI, and in contrast to that provoked by PARs, involves tyrosine phosphorylation of the adaptor protein LAT as well as of PLCγ2 in a src- and Syk-dependent manner. In this respect, we provide evidence that Pam3CSK4 does not cross-activate GPVI.Further, by the use of platelets from a Glanzmann's thrombasthenia patient lacking β₃, in contrast to findings in nucleated immune cells, we show that the initiation of platelet activation by Pam3CSK4 does not involve integrin β₃ signalling; whereas the latter, subsequent to intermediate TXA2 synthesis and signalling, was found to be indispensable for proper dense granule secretion and full platelet aggregation. Together, our findings reveal that triggering the TLR2/1 complex with Pam3CSK4 initiates human platelet activation by engaging tyrosine kinases of the src family and Syk, the adaptor protein LAT, as well as the key mediator PLCγ2.     

On the solvatochromic properties of the oxalate-bridged complexes [(BuCO2)3M2]2(μ-O2C2O2) where M = Mo or W

The room temperature electronic absorption spectra of the oxalate bridged MM quadruply bonded complexes [(^tBuCO₂)₃M₂]₂(μ-O₂C₂O₂), where M = Mo or W have been recorded in H₂O, THF:H₂O mixtures, THF, CH₂Cl₂, toluene, DMSO, aniline, toluene saturated with N,N-dimethylaniline and ethanol. The strong absorptions in the visible region of the electronic absorption spectra assignable to the metal-to-ligand (bridge) charge transfer are shown to be highly solvent dependent. Those samples prepared in H₂O, CH₂Cl₂ and toluene are shown to comprise of a suspension of microcrystalline particles ranging in size from 100 nm to 5 μm. Individual particles were found by scanning electron microscopy to have an aspect ratio of ∼10:1, all being needle shaped. The spectra in THF, EtOH, aniline, DMSO and toluene-N,N-dimethylaniline all show similar vibronic progressions and are attributed to discrete solvated molecular species. The spectra recorded in aniline are notably red-shifted which is proposed to arise from a combination of hydrogen bonding and Lewis base stabilization of the photoexcited state.     

Direct Interactions with the Integrin β1 Cytoplasmic Tail Activate the Abl2/Arg Kinase

Integrins are heterodimeric α/β extracellular matrix adhesion receptors that couple physically to the actin cytoskeleton and regulate kinase signaling pathways to control cytoskeletal remodeling and adhesion complex formation and disassembly. β1 integrins signal through the Abl2/Arg (Abl-related gene) nonreceptor tyrosine kinase to control fibroblast cell motility, neuronal dendrite morphogenesis and stability, and cancer cell invasiveness, but the molecular mechanisms by which integrin β1 activates Arg are unknown. We report here that the Arg kinase domain interacts directly with a lysine-rich membrane-proximal segment in the integrin β1 cytoplasmic tail, that Arg phosphorylates the membrane-proximal Tyr-783 in the β1 tail, and that the Arg Src homology domain then engages this phosphorylated region in the tail. We show that these interactions mediate direct binding between integrin β1 and Arg in vitro and in cells and activate Arg kinase activity. These findings provide a model for understanding how β1-containing integrins interact with and activate Abl family kinases.     

Synthesis and structural characterization of the molecular loop [cis-Mo2(N,N-di-p-anisylformamidinate)2]2(O2C-CHCCH-CO2)2

A new molecular loop composed of two quadruply bonded Mo₂(DAniF)₂ units (DAniF=N,N^′-di-p-anisylformamidinate) linked by two chiral allene-1,3-dicarboxylate anions has been prepared from the reaction of [cis-Mo₂(DAniF)₂(MeCN)₄](BF₄)₂ with the bis(tetraethylammonium) salt of allene-1,3-dicarboxylic acid. This compound, [cis-Mo₂(DAniF)₂]₂(O₂C-CHCCH-CO₂)₂ (1), has been characterized by X-ray crystallography and by ¹H NMR and UV-Vis spectroscopy. The molecule possesses a center of inversion and hence is meso. There is only weak electronic coupling between the two Mo₂ ⁴⁺ units as revealed by electrochemical measurements.     

Physical and functional interaction of the active zone protein CAST/ERC2 and the β-subunit of the voltage-dependent Ca(2+) channel

In the nerve terminals, the active zone protein CAST/ERC2 forms a protein complex with the other active zone proteins ELKS, Bassoon, Piccolo, RIM1 and Munc13-1, and is thought to play an organizational and functional role in neurotransmitter release. However, it remains obscure how CAST/ERC2 regulates the Ca(2+)-dependent release of neurotransmitters. Here, we show an interaction of CAST with voltage-dependent Ca(2+) channels (VDCCs), which are essential for regulating neurotransmitter release triggered by depolarization-induced Ca(2+) influx at the active zone. Using a biochemical assay, we showed that CAST was coimmunoprecipitated with the VDCC β(4)-subunit from the mouse brain. A pull-down assay revealed that the VDCC β(4)-subunit interacted directly with at least the N- and C-terminal regions of CAST. The II-III linker of VDCC α(1)-subunit also interacted with C-terminal regions of CAST; however, the interaction was much weaker than that of β(4)-subunit. Furthermore, coexpression of CAST and VDCCs in baby hamster kidney cells caused a shift in the voltage dependence of activation towards the hyperpolarizing direction. Taken together, these results suggest that CAST forms a protein complex with VDCCs, which may regulate neurotransmitter release partly through modifying the opening of VDCCs at the presynaptic active zones.     

Allosteric interactions between the oxytocin receptor and the β2-adrenergic receptor in the modulation of ERK1/2 activation are mediated by heterodimerization

The oxytocin receptor (OTR) and the β₂-adrenergic receptor (β₂AR) are key regulators of uterine contraction. These two receptors are targets of tocolytic agents used to inhibit pre-term labor. Our recent study on the nature of OTR- and β₂AR-mediated ERK1/2 activation in human hTERT-C3 myometrial cells suggested the presence of an OTR/β₂AR hetero-oligomeric complex (see companion article). The goal of this study was to investigate potential allosteric interactions between OTR and β₂AR and establish the nature of the interactions between these receptors in myometrial cells. We found that OTR-mediated ERK1/2 activation was attenuated significantly when cells were pretreated with the β₂AR agonist isoproterenol or two antagonists, propranolol or timolol. In contrast, pretreatment of cells with a third β₂AR antagonist, atenolol resulted in an increase in OTR-mediated ERK1/2 activation. Similarly, β₂AR-mediated ERK1/2 activation was strongly attenuated by pretreatment with the OTR antagonists, atosiban and OTA. Physical interactions between OTR and β₂AR were demonstrated using co-immunoprecipitation, bioluminescence resonance energy transfer (BRET) and protein-fragment complementation (PCA) assays in HEK 293 cells, the latter experiments indicating the interactions between the two receptors were direct. Our analyses suggest physical interactions between OTR and β₂AR in the context of a new heterodimer pair lie at the heart of the allosteric effects.     

Dynamic relocation of the TORC1–Gtr1/2–Ego1/2/3 complex is regulated by Gtr1 and Gtr2

TORC1 regulates cellular growth, metabolism, and autophagy by integrating various signals, including nutrient availability, through the small GTPases RagA/B/C/D in mammals and Gtr1/2 in budding yeast. Rag/Gtr is anchored to the lysosomal/vacuolar membrane by the scaffold protein complex Ragulator/Ego. Here we show that Ego consists of Ego1 and Ego3, and novel subunit Ego2. The ∆ego2 mutant exhibited only partial defects both in Gtr1-dependent TORC1 activation and Gtr1 localization on the vacuole. Ego1/2/3, Gtr1/2, and Tor1/Tco89 were colocalized on the vacuole and associated puncta. When Gtr1 was in its GTP-bound form and TORC1 was active, these proteins were preferentially localized on the vacuolar membrane, whereas when Gtr1 was in its GDP-bound form, they were mostly localized on the puncta. The localization of TORC1 to puncta was further facilitated by direct binding to Gtr2, which is involved in suppression of TORC1 activity. Thus regulation of TORC1 activity through Gtr1/Gtr2 is tightly coupled to the dynamic relocation of these proteins.     

Desipramine selectively potentiates norepinephrine-elicited ERK1/2 activation through the α2A adrenergic receptor

The precise physiological effects of antidepressant drugs, and in particular their actions at non-monoamine transporter targets, are largely unknown. We have recently identified the tricyclic antidepressant drug desipramine (DMI) as a direct ligand at the α(2A) adrenergic receptor (AR) without itself driving heterotrimeric G protein/downstream effector activation [5]. In this study, we report our novel finding that DMI modulates α(2A)AR signaling in response to the endogenous agonist norepinephrine (NE). DMI acted as a signaling potentiator, selectively enhancing NE-induced α(2A)AR-mediated ERK1/2 MAPK signaling. This potentiation of ERK1/2 activation was observed as an increase in NE response sensitivity and a prolongation of the activation kinetics. DMI in a physiologically relevant ratio with NE effectively turned on ERK1/2 signaling that is lacking in response to physiological NE alone. Further, the DMI-induced ERK1/2 potentiation relied on heterotrimeric G(i/o) proteins and was arrestin-independent. This modulatory effect of DMI on NE signaling provides novel insight into the effects of this antidepressant drug on the noradrenergic system which it regulates, insight which enhances our understanding of the therapeutic mechanism for DMI.     

Inhibitors of the p53/hdm2 protein–protein interaction—path to the clinic

A growing number of the elements identified in intracellular signaling events that affect cell growth and transformation are proteins that physically interact with each other via domains or specifically recognized amino acid sequences. Some of these intracellular protein–protein interactions are attractive targets for anticancer targeted therapy, but progress in this field has been compromised by the paucity of compounds with suitable biological profiles and pharmacological properties. This Letter covers salient achievements in the identification and development of inhibitors of the p53–hdm2 protein–protein interaction, and highlights different screening techniques and structure-based design approaches that may be brought to bear on the discovery and development of inhibitors of other therapeutically relevant intracellular protein–protein interactions.     

Comparison of the human genomic structure of the Runt domain-encoding PEBP2/CBFα gene family

We cloned the human cDNA corresponding to the cDNA (PEBP2αB-451) encoding the mouse polyoma virus enhancer-binding protein 2αtB-451, representing a major splice variant from acute myeloid leukemia gene 1 (AMLI). Genomic DNA clones of AMLI were also isolated and the exon/intron structure was determined. Furthermore, we determined and compared the genomic structures of three mammalian Runt domain-containing genes, PEBP2αA, AMLI/PEBP2αB and PEBP2αC.     

Role of the nitric oxide/cyclic GMP/Ca2+ signaling pathway in the pyrogenic effect of interleukin-1β

Interleukin-1β (IL-1β) has a wide spectrum of inflammatory, metabolic, haemopoietic, and immunological properties. Because it produces fever when injected into animals and humans, it is considered an endogenous pyrogen. There is evidence to suggest that Ca²⁺ plays a critical role in the central mechanisms of thermoregulation, and in the intracellular signaling pathways controlling fever induced by IL-1β and other pyrogens. Data from different labs indicate that Ca²⁺ and Na⁺ determine the temperature set point in the posterior hypothalamus (PH) of various mammals and that changes in Ca²⁺ and PGE₂ concentrations in the cerebrospinal fluid (CSF) of these animals are associated with IL-1β-induced fever. Antipyretic drugs such as acetylsalicylic acid, dexamethasone, and lipocortin 5-(204–212) peptide counteract IL-1β-induced fever and abolish changes in Ca²⁺ and PGE₂ concentrations in CSF. In vitro studies have established that activation of the nitric oxide (NO)/cyclic GMP (cGMP) pathway is part of the signaling cascade transducing Ca²⁺ mobilization in response to IL-1β and that the ryanodine (RY)- and inositol-(1,4,5)-trisphosphate (IP₃)-sensitive pools are the main source of the mobilized Ca²⁺. It is concluded that the NO/cGMP/Ca²⁺ pathway is part of the signaling cascade subserving some of the multiple functions of IL-1β.