ABSTRACT
Lipid raft nanodomains of plasma membrane are rich in saturated lipids‚ cholesterol‚ sphingolipids‚ functioning as multimolecular platforms to recruit signaling and trafficking proteins involved in an array of physiological processes‚ which are critical for regulating signal transduction in cell. The staggering complexity of cell membranes and the transient formation of nanodomains greatly hinder research on lipid rafts by traditional experimental means. Molecular dynamics simulations have provided important insight into the organizational principles of cell membranes recently. Simulated membrane systems are under a transition from simple membrane models to multicomponent systems‚ culminating in realistic models of various cell types. Coarse-grained models have been extensively adopted as a powerful tool to explore membrane organization and interactions between lipids and proteins‚ providing efficient computational speed and enabling complex systems. In this work‚ coarse-grained molecular dynamics simulations with MARTINI force field were performed to build a raft-forming membrane with mixed lipids‚ including negatively charged lipid PIP2. Mixed lipids in this model were spontaneously partitioned into binary-phase membrane during 5 μs simulations by low temperature (295 K) treatment‚ forming lipid ordered (Lo) and liquid-disordered (Ld) nanodomains. Results of membrane thickness‚ lipid distribution‚ membrane fluidity‚ order parameters of the acyl tails‚ radial distribution functions were consistent with simulation and experimental data. Addition of small amounts of PIP2 did not affect the raft formation‚ and it showed remarkable affinity to lipid raft nanodomains. Simulations of the signaling transmembrane protein CD3ε in our raft-forming membranes were further performed to study the protein-lipid interaction as well. Results showed that the cytoplasmic tail of CD3ε was recruited to the Lo/ Ld boundary due to PIP2 binding‚ and this binding was regulated by Ca
ABSTRACT
Tor2 is an activator of the Rom2/Rho1 pathway that regulates α-factor internalization. Since the recruitment of endocytic proteins such as actin-binding proteins and the amphiphysins precedes the internalization of α-factor, we hypothesized that loss of Tor function leads to an alteration in the dynamics of the endocytic proteins. We report here that endocytic proteins, Abp1 and Rvs167, are less recruited to endocytic sites not only in tor2 but also tor1 mutants. Furthermore, we found that the endocytic proteins Rvs167 and Sjl2 are completely mistargeted to the cytoplasm in tor1Δtor2ts double mutant cells. We also demonstrate here that the efficiency of endocytic internalization or scission in all tor mutants was drastically decreased. In agreement with the Sjl2 mislocalization, we found that in tor1Δtor2ts double mutant cells, as well as other tor mutant cells, the overall PIP2 level was dramatically increased. Finally, the cell wall chitin content in tor2ts and tor1Δtor2ts mutant cells was also significantly increased. Taken together, both functional Tor proteins, Tor1 and Tor2, are essentially required for proper endocytic protein dynamics at the early stage of endocytosis.
ABSTRACT
Objective To explore the role of PLC/PIP2 signal pathway in the changes of mouse thymus CD4 + CD25 + NRP1 + Treg and TGF-β1 after different doses of X-ray irradiation Methods 36 ICR mice were randomly divided into 6 groups according to the irradiation doses of 0,0.5,1.0,2.0,4.0 and 6.0 Gy,respectively.Flow cytometry was used to detect the expression of NRP1 + Treg,and ELISA was used to detect the expression of TGF-β1 in mouse thymocytes at 16 h post-irradiation.The EL-4 cells were irradiated by X-rays at the dose of 4.0 Gy after co-cultured with the PMA and TMB-8 for 2 hours.Flow cytometry was used to detect the expression of NRP1 + Treg,and ELISA was used to detect the changes of TGF-β1 at 48 h post-irradiation.Results The NRP1 + Treg appeared a transient decrease at both 0.5 and 1.0 Gy irradiation and reached its valley value at 1.0 Gy (t =6.96,P ＜ 0.01),then showed a dosedependent increase and reached its peak at 6.0 Gy (t =6.70,P ＜ 0.01).The TGF-β1 level decreased after 0.5 Gy X-rays (t =12.53,P ＜0.01),then increased at a dose-dependent manner and reached its peak at 4.0 Gy (t =10.40-19.56,P ＜ 0.01).Compared with the sham-irradiation,NRP1 + Treg was decreased significantly after PMA treatment (t =3.06,P ＜ 0.01),while it was up-regulated significantly after irradiation in the presence of PMA (t =8.27,P ＜ 0.01).TGF-β1 was reduced in the presence of PMA with or without irradiation (t =10.46-39.69,P ＜ 0.01).NRP1 + Treg and TGF-β1 were increased significantly after TMB-8 treatment (t =5.53-44.26,P ＜ 0.01).Conclusions NRP1 + Treg cells and TGF-β1 were up-regulated after a high dose radiation,and the PLC/PIP2 signal pathway may participate in the regulation.
ABSTRACT
Adenosine (Ado) is an important mediator of the endogenous defense against ischemia-induced injury in the heart. The action of Ado is mediated by activation of G protein-gated inwardly rectifying K+ (GIRK) channels. In turn, GIRK channels are inhibited by reducing phosphatidylinositol 4,5-bisphosphate (PIP2) through Gq protein-coupled receptors (GqPCRs). We previously found that GIRK channels activated by acetylcholine, a muscarinic M2 acetylcholine receptor agonist, are inhibited by GqPCRs in a receptor-specific manner. However, it is not known whether GIRK channels activated by Ado signaling are also regulated by GqPCRs. Presently, this was investigated in mouse atrial myocytes using the patch clamp technique. GIRK channels were activated by 100 micrometer Ado. When Ado was repetitively applied at intervals of 5~6 min, the amplitude of second Ado-activated GIRK currents (I(K(Ado))) was 88.3+/-3.7% of the first I(K(Ado)) in the control. Pretreatment of atrial myocytes with phenylephrine, endothelin-1, or bradykinin prior to a second application of Ado reduced the amplitude of the second I(K(Ado)) to 25.5+/-11.6%, 30.5+/-5.6%, and 96.0+/-2.7%, respectively. The potency of I(K(Ado)) inhibition by GqPCRs was different with that observed in acetylcholine-activated GIRK currents (I(K(ACh))) (endothelin-1>phenylephrine>bradykinin). I(K(Ado)) was almost completely inhibited by 500 micrometer of the PIP2 scavenger neomycin, suggesting low PIP2 affinity of I(K(Ado)). Taken together, these results suggest that the crosstalk between GqPCRs and the Ado-induced signaling pathway is receptor-specific. The differential change in PIP2 affinity of GIRK channels activated by Ado and ACh may underlie, at least in part, their differential responses to GqPCR agonists.
Subject(s)
Animals , Mice , Acetylcholine , Adenosine , Bradykinin , Carotenoids , Endothelin-1 , Heart , Muscle Cells , Neomycin , Oxygenases , Phenylephrine , PhosphatidylinositolsABSTRACT
Aim To study the regulatory effects of PMA,a PKC activator,on Kir 2.3 channel function expressed in Xenopus oocytes and COS-7 cells,and PIP2 involvement in these regulations.Methods Kir 2.3 channel was expressed in Xenopus oocytes and COS-7 cell by RNA microinjection and DNA transfection using calcium phosphate precipitate,respectively.Two-electrode-voltage-clamp and whole-cell patch clamp were used to record the Kir 2.3 current in Xenopus oocytes and COS-7 cell.The PIP2 hydrolysis was detected by confocal microscopy.Results PMA significantly inhibited Kir 2.3 current in Xenopus oocytes.But PMA had no effect on the Kir 2.3 current expressed in COS-7 cell,in which activation of M1 receptor,however,induced a significant inhibition of Kir 2.3 current.It was reported recently that PMA could trigger the PIP2 hydrolysis in membrane of oocytes.Thus PKC inhibition of Kir 2.3 current seen in oocytes could be the result of PIP2 hydrolysis.Following the same line,the inability of PKC inhibition of Kir 2.3 current seen in COS-7 cells would suggest PKC could not induce PIP2 hydrolysis in these cells. This hypothesis was tested by monitoring the PIP2 level in COS-7 cell membrane by confocal microscopy.Dynamic changes in membrane PIP2 level were imaged using GFP fluorescence signal that had been tagged to the PLC?1PH domain known to be able to bind PIP2 specifically. There was no significant change of PIP2 level on COS-7 cell membrane after longtime treatment of PMA,whereas again,the activation of M1 receptor by ACh induced a significant change in the PIP2 level.These results were in perfect agreement with the electrophysiological results.Conclusions PMA,through activation of PKC,inhibited Kir 2.3 current expressed in Xenopus oocytes but not in COS-7 cells.Similarly PMA induced significant reduction in membrane PIP2 level in Xenopus oocytes but not in COS-7 cells. PIP2 hydrolysis plays an important role in PKC-induced inhibition of the Kir channel currents.
ABSTRACT
As an outward,voltage-dependent potassium channel,M type channel is crucial in the regulation of neuronal excitability;it is modulated by a variety of factors in vivo and its dysfunction often results in neuronal system diseases.Great efforts have been made to elucidate the mechanism underlying M channel modulation since its discovery decades ago.It is generally accepted that the Phospholipase C(PLC) signaling pathway plays a significant role in the M channel modulation.This review highlights the relationship between PLC signaling pathway and M channel modulation,as well as some recent progresses in the research of this field.
ABSTRACT
To investigate dynamic characteristics of the hydrolization lipid phosphatidylinositol(4,5)-bisphosphate(PIP 2 )with the stimulation of epidermal growth factor(EGF),the mathematical model to simulate the metabolizability of PIP 2 is established based on Law of Mass Action and Law of the Quasi-steady-State Approximation.Differential equations of concentration relations between PIP 2 and EGF receptor are formulated,and the effect of the parameters on the changing trend of PIP 2 is analyzed.This mathematical model describes biology characteristics of metabolized PIP 2 and dependency relationships of concentration between key signal producuts.