Lia Baki

Presenilin-1 binds cytoplasmic epithelial cadherin, inhibits cadherin/p120 association, and regulates stability and function of the cadherin/catenin adhesion complex

Here we show that presenilin-1 (PS1), a protein involved in Alzheimers disease, binds directly to epithelial cadherin (E-cadherin). This binding is mediated by the large cytoplasmic loop of PS1 and requires the membrane-proximal cytoplasmic sequence 604–615 of mature E-cadherin. This sequence is also required for E-cadherin binding of protein p120, a known regulator of cadherin-mediated cell adhesion. Using wild-type and PS1 knockout cells, we found that increasing PS1 levels suppresses p120/E-cadherin binding, and increasing p120 levels suppresses PS1/E-cadherin binding. Thus PS1 and p120 bind to and mutually compete for cellular E-cadherin. Furthermore, PS1 stimulates E-cadherin binding to β- and γ-catenin, promotes cytoskeletal association of the cadherin/catenin complexes, and increases Ca2+-dependent cell–cell aggregation. Remarkably, PS1 familial Alzheimer disease mutant ΔE9 increased neither the levels of cadherin/catenin complexes nor cell aggregation, suggesting that this familial Alzheimer disease mutation interferes with cadherin-based cell–cell adhesion. These data identify PS1 as an E-cadherin-binding protein and a regulator of E-cadherin function in vivo.

Wild-Type But Not FAD Mutant Presenilin-1 Prevents Neuronal Degeneration by Promoting Phosphatidylinositol 3-Kinase Neuroprotective Signaling

The role of presenilin-1 (PS1) in neuronal phosphatidylinositol 3-kinase (PI3K)/Akt signaling was investigated in primary neuronal cultures from wild-type (WT) and PS1 null (PS1−/−) embryonic mouse brains. Here we show that in PS1−/− cultures, the onset of neuronal maturation coincides with a decrease in the PI3K-dependent phosphorylation-activation of Akt and phosphorylation-inactivation of glycogen synthase kinase-3 (GSK-3). Mature PS1−/− neurons show increased activation of apoptotic caspase-3 and progressive degeneration preceded by dendritic retraction. Expression of exogenous WT PS1 or constitutively active Akt in PS1−/− neurons stimulates PI3K signaling and suppresses both caspase-3 activity and dendrite retraction. The survival effects of PS1 are sensitive to inhibitors of PI3K kinase but insensitive to γ-secretase inhibitors. Familial Alzheimer disease (FAD) mutations suppress the ability of PS1 to promote PI3K/AKT signaling, prevent phosphorylation/inactivation of GSK-3 and promote activation of caspase-3. These mutation effects are reversed upon coexpression of constitutively active Akt. Together, our data indicate that the neuroprotective role of PS1 depends on its ability to activate the PI3K/Akt signaling pathway and that PS1 FAD mutations increase GSK-3 activity and promote neuronal apoptosis by inhibiting the function of PS1 in this pathway. These observations suggest that stimulation of PI3K/Akt signaling may be beneficial to FAD patients.

Phosphatidylinositol-4,5-bisphosphate regulates epidermal growth factor receptor activation

Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2 or PIP2] is a direct modulator of a diverse array of proteins in eukaryotic cells. The functional integrity of transmembrane proteins, such as ion channels and transporters, is critically dependent on specific interactions with PIP2 and other phosphoinositides. Here, we report a novel requirement for PIP2 in the activation of the epidermal growth factor receptor (EGFR). Down-regulation of PIP2 levels either via pharmacological inhibition of PI kinase activity, or via manipulation of the levels of the lipid kinase PIP5K1α and the lipid phosphatase synaptojanin, reduced EGFR tyrosine phosphorylation, whereas up-regulation of PIP2 levels via overexpression of PIP5K1α had the opposite effect. A cluster of positively charged residues in the juxtamembrane domain (basic JD) of EGFR is likely to mediate binding of EGFR to PIP2 and PIP2-dependent regulation of EGFR activation. A peptide mimicking the EGFR juxtamembrane domain that was assayed by surface plasmon resonance displayed strong binding to PIP2. Neutralization of positively charged amino acids abolished EGFR/PIP2 interaction in the context of this peptide and down-regulated epidermal growth factor (EGF)-induced EGFR auto-phosphorylation and EGF-induced EGFR signaling to ion channels in the context of the full-length receptor. These results suggest that EGFR activation and downstream signaling depend on interactions of EGFR with PIP2 and point to the basic JD’s critical involvement in these interactions. The addition of this very different class of membrane proteins to ion channels and transporters suggests that PIP2 may serve as a general modulator of the activity of many diverse eukaryotic transmembrane proteins through their basic JDs.

Phosphoinositide Control of Membrane Protein Function: A Frontier Led by Studies on Ion Channels

Anionic phospholipids are critical constituents of the inner leaflet of the plasma membrane, ensuring appropriate membrane topology of transmembrane proteins. Additionally, in eukaryotes, the negatively charged phosphoinositides serve as key signals not only through their hydrolysis products but also through direct control of transmembrane protein function. Direct phosphoinositide control of the activity of ion channels and transporters has been the most convincing case of the critical importance of phospholipid-protein interactions in the functional control of membrane proteins. Furthermore, second messengers, such as [Ca(2+)]i, or posttranslational modifications, such as phosphorylation, can directly or allosterically fine-tune phospholipid-protein interactions and modulate activity. Recent advances in structure determination of membrane proteins have allowed investigators to obtain complexes of ion channels with phosphoinositides and to use computational and experimental approaches to probe the dynamic mechanisms by which lipid-protein interactions control active and inactive protein states.

Allosteric signaling through an mGlu2 and 5-HT2A heteromeric receptor complex and its potential contribution to schizophrenia

Stimulation of one partner in a heteromeric GPCR complex in the brain activates the other partner. One to bind, one to signal In addition to forming homodimers and heterodimers, G protein–coupled receptors (GPCRs) can form multiprotein complexes (heteromers) with other GPCRs. For example, the metabotropic glutamate receptor mGlu2, which couples to Gi/o proteins, and the 5-HT2A serotonin receptor, which couples to Gq/11, form heteromeric complexes. Moreno et al. performed a structure-function analysis to determine the signaling properties of these heteromers. Stimulation of cells expressing mGlu2–5-HT2A heteromers with an mGlu2 agonist led to Gq/11-dependent signaling by 5-HT2A, a response lacking in cells from 5-HT2A–deficient mice. Furthermore, the analysis of postmortem brains of schizophrenia patients indicated less mGlu2-dependent Gq/11 signaling compared to that in normal brains, suggesting that these heteromeric complexes may be dysregulated in disease. Heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) can form multiprotein complexes (heteromers), which can alter the pharmacology and functions of the constituent receptors. Previous findings demonstrated that the Gq/11-coupled serotonin 5-HT2A receptor and the Gi/o-coupled metabotropic glutamate 2 (mGlu2) receptor—GPCRs that are involved in signaling alterations associated with psychosis—assemble into a heteromeric complex in the mammalian brain. In single-cell experiments with various mutant versions of the mGlu2 receptor, we showed that stimulation of cells expressing mGlu2–5-HT2A heteromers with an mGlu2 agonist led to activation of Gq/11 proteins by the 5-HT2A receptors. For this crosstalk to occur, one of the mGlu2 subunits had to couple to Gi/o proteins, and we determined the relative location of the Gi/o-contacting subunit within the mGlu2 homodimer of the heteromeric complex. Additionally, mGlu2-dependent activation of Gq/11, but not Gi/o, was reduced in the frontal cortex of 5-HT2A knockout mice and was reduced in the frontal cortex of postmortem brains from schizophrenic patients. These findings offer structural insights into this important target in molecular psychiatry.

Gating of a G protein-sensitive Mammalian Kir3.1 Prokaryotic Kir Channel Chimera in Planar Lipid Bilayers

Kir3 channels control heart rate and neuronal excitability through GTP-binding (G) protein and phosphoinositide signaling pathways. These channels were the first characterized effectors of the βγ subunits of G proteins. Because we currently lack structures of complexes between G proteins and Kir3 channels, their interactions leading to modulation of channel function are not well understood. The recent crystal structure of a chimera between the cytosolic domain of a mammalian Kir3.1 and the transmembrane region of a prokaryotic KirBac1.3 (Kir3.1 chimera) has provided invaluable structural insight. However, it was not known whether this chimera could form functional K+ channels. Here, we achieved the functional reconstitution of purified Kir3.1 chimera in planar lipid bilayers. The chimera behaved like a bona fide Kir channel displaying an absolute requirement for PIP2 and Mg2+-dependent inward rectification. The channel could also be blocked by external tertiapin Q. The three-dimensional reconstruction of the chimera by single particle electron microscopy revealed a structure consistent with the crystal structure. Channel activity could be stimulated by ethanol and activated G proteins. Remarkably, the presence of both activated Gα and Gβγ subunits was required for gating of the channel. These results confirm the Kir3.1 chimera as a valid structural and functional model of Kir3 channels.

Cholesterol increases the open probability of cardiac KACh currents

Cholesterol is one of the major lipid components of membranes in mammalian cells. In recent years, cholesterol has emerged as a major regulator of ion channel function. The most common effect of cholesterol on ion channels in general and on inwardly rectifying potassium (Kir) channels in particular is a decrease in activity. In contrast, we have recently shown that native G-protein gated Kir (GIRK or Kir3) channels that underlie atrial KACh currents are up-regulated by cholesterol. Here we unveil the biophysical basis of cholesterol-induced increase in KACh activity. Using planar lipid bilayers we show that cholesterol significantly enhances the channel open frequency of the Kir3.1/Kir3.4 channels, which underlie KACh currents. In contrast, our data indicate that cholesterol does not affect their unitary conductance. Furthermore, using fluorescent and TIRF microscopy as well as surface protein biotinylation, we also show that cholesterol enrichment in vitro has no effect on surface expression of GFP-tagged channels expressed in Xenopus oocytes or transfected into HEK293 cells. Together, these data demonstrate for the first time that cholesterol enhances Kir3-mediated current by increasing the channel open probability.

Reformulating a Pharmacophore for 5-HT2A Serotonin Receptor Antagonists

Several pharmacophore models have been proposed for 5-HT2A serotonin receptor antagonists. These typically consist of two aromatic/hydrophobic moieties separated by a given distance from each other, and from a basic amine. Although specified distances might vary, the models are relatively similar in their general construction. Because our preliminary data indicated that two aromatic (hydrophobic) moieties might not be required for such action, we deconstructed the serotonin-dopamine antipsychotic agent risperidone (1) into four smaller structural fragments that were thoroughly examined in 5-HT2A receptor binding and functional (i.e., two-electrode voltage clamp (TEVC) and intracellular calcium release) assays. It was apparent that truncated risperidone analogues behaved as antagonists. In particular, 6-fluoro-3-(1-methylpiperidin-4-yl)benzisoxazole (4) displayed high affinity for 5-HT2A receptors (Ki of ca. 12 nM) relative to risperidone (Ki of ca. 5 nM) and behaved as a potent 5-HT2A serotonin receptor antagonist. These results suggest that multiple aromatic (hydrophobic) moieties are not essential for high-affinity 5-HT2A receptor binding and antagonist activity and that current pharmacophore models for such agents are very much in need of revision.

Pronounced enhancement of glucocorticoid-induced gene expression following severe heat shock of heat-conditioned cells hints to intricate cell survival tactics

We have previously reported that severe heat shock of HeLa cells stably transfected with a chloramphenicol acetyltransferase (CAT) gene, transcription of which is controlled by two glucocorticoid-responsive elements and a minimal promoter, pronouncedly enhanced glucocorticoid-induced CAT expression compared to that of non-heated cells, in spite of the glucocorticoid-receptor-mediated transcription of the gene being temporarily compromised by the shock. We now report that prolonged severe heat shock of properly heat-conditioned cells resulted in far more pronounced enhancement of glucocorticoid-induced CAT mRNA and protein expressions, in spite of a similar heat-induced loss of receptor-mediated CAT gene transcription. During recovery from the shock the hormonal activation of transcription exceeded that of non-heated cells. While CAT mRNA translation was restored appreciably later than CAT gene transcription, mRNA and protein expressions were thermally enhanced to a comparable extent, consistent with the integrity of CAT mRNA being preserved during recovery. CAT mRNA turnover was fully impaired during early recovery, suggesting that stabilisation of CAT mRNA as well as stimulation of the hormonal activation of CAT gene transcription account for the thermal enhancement of glucocorticoid-induced CAT expression. This data hint to cell survival tactics designed to safeguard high expression of genes of stress-enduring function.