Opioid-induced proinflammatory glial activation modulates wide-ranging aspects of opioid pharmacology including: opposition of acute and chronic opioid analgesia, opioid analgesic tolerance, opioid-induced hyperalgesia, development of opioid dependence, opioid reward, and opioid respiratory depression. However, the mechanism(s) contributing to opioid-induced proinflammatory actions remains unresolved. The potential involvement of toll like receptor 4 (TLR4) was examined using in vitro, in vivo, and in silico techniques. Morphine non-stereoselectively induced TLR4 signaling in vitro, blocked by a classical TLR4 antagonist and non-stereoselectively by naloxone. Pharmacological blockade of TLR4 signaling in vivo potentiated acute intrathecal morphine analgesia, attenuated development of analgesic tolerance, hyperalgesia, and opioid withdrawal behaviors. TLR4 opposition to opioid actions was supported by morphine treatment of TLR4 knockout mice, which revealed a significant threefold leftward shift in the analgesia dose response function, versus wildtype mice. A range of structurally diverse clinically employed opioid analgesics was found to be capable of activating TLR4 signaling in vitro. Selectivity in the response was identified since morphine-3-glucuronide, a morphine metabolite with no opioid receptor activity, displayed significant TLR4 activity, whilst the opioid receptor active metabolite, morphine-6-glucuronide, was devoid of such properties. In silico docking simulations revealed ligands bound Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptBrain Behav Immun. Author manuscript; available in PMC 2011 January 1. preferentially to the LPS binding pocket of MD-2 rather than TLR4. An in silico to in vitro prediction model was built and tested with substantial accuracy. These data provide evidence that select opioids may non-stereoselectively influence TLR4 signaling and have behavioral consequences resulting, in part, via TLR4 signaling.

The protein kinase AKT1 regulates multiple signaling pathways essential for cell function. Its Nterminal PH domain (AKT1 PH) binds the rare signaling phospholipid phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ], resulting in plasma membrane targeting and phosphoactivation of AKT1 by a membrane-bound kinase. Recently, it was discovered that the Glu17Lys mutation in the AKT1 PH domain is associated with multiple human cancers. This mutation constitutively targets the AKT1 PH domain to the plasma membrane by an unknown mechanism, thereby promoting constitutive AKT1 activation and oncogenesis. To elucidate the molecular mechanism underlying constitutive plasma membrane targeting, this work compares the membrane docking reactions of the isolated wild-type and E17K AKT1 PH domains. In vitro studies reveal that the E17K mutation dramatically increases the affinity for the constitutive plasma membrane lipid PI(4,5)P 2 . The resulting PI(4,5)P 2 equilibrium affinity is indistinguishable from that of the standard PI(4,5)P 2 sensor, PLCδ1 PH domain. Kinetic studies indicate that the effects of E17K on PIP lipid binding arise largely from electrostatic modulation of the dissociation rate. Membrane targeting analysis in live cells confirms that the constitutive targeting of E17K AKT1 PH to plasma membrane, like PLCδ1 PH, stems from PI(4,5)P 2 binding. Overall, the evidence indicates that the molecular mechanism underlying E17K oncogenesis is a broadened target lipid selectivity that allows high-affinity binding to PI(4,5)P 2 . Moreover, the findings strongly implicate the native Glu17 side chain as a key element of PIP lipid specificity in the wild-type AKT1 PH domain. Other PH domains may employ an analogous anionic residue to control PIP specificity.Many cellular signaling processes are controlled by events occurring at the surface of intracellular membranes. Phosphatidylinositiol phosphates (PIPs) comprise an important class of signaling phospholipids that are highly regulated second messengers and can drive the reversible localization of key signaling proteins from the cytoplasm to the inner leaflet of the plasma membrane (1-5). Such PIP-dependent localization events play essential roles in many signaling pathways. For example, on the cytoplasmic surface of the plasma membrane, the phosphatidylinositol 3-kinase (PI3K) family of lipid kinases convert a constitutive PIP lipid, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ], 1 into the rare second-messenger lipid phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ] (1,6,7). This second messenger, in turn, recruits a wide array of important downstream protein effectors † Supported by NIH Grant R01 GM-063235 (to J.J. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 August 10. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript containing pleckstrin homology (PH) domains that specifically bind to the PI(3,4,5)P 3 headgroup (8-10). The resulting plasma membrane targeting activates signali...

The C2 domain is a ubiquitous, conserved protein signaling motif widely found in eukaryotic signaling proteins. Although considerable functional diversity exists, most C2 domains are activated by Ca 2+ binding and then dock to a specific cellular membrane. The C2 domains of protein kinase Cα (PKCα) and cytosolic phospholipase A 2 α (cPLA 2 α), for example, are known to dock to different membrane surfaces during an intracellular Ca 2+ signal. Ca 2+ activation targets the PKCα C2 domain to the plasma membrane and the cPLA 2 α C2 domain to the internal membranes, with no detectable spatial overlap. It is crucial to determine how such targeting specificity is achieved at physiological bulk Ca 2+ concentrations that during a typical signaling event rarely exceed 1 μM. For the isolated PKCα C2 domain in the presence of physiological Ca 2+ levels, the target lipids phosphatidylserine (PS) and phosphatidylinositol-4,5-bisphosphate (PIP 2 ) are together sufficient to recruit the PKCα C2 domain to a lipid mixture mimicking the plasma membrane inner leaflet. For the cPLA 2 α C2 domain, the target lipid phosphatidylcholine (PC) appears to be sufficient to drive membrane targeting to an internal membrane mimic at physiological Ca 2+ levels, although the results do not rule out a second, unknown target molecule. Stopped-flow kinetic studies provide additional information about the fundamental molecular events that occur during Ca 2+ -activated membrane docking. In principle, C2 domain-directed intracellular targeting, which requires coincidence detection of multiple signals (Ca 2+ and one or more target lipids), can exhibit two different mechanisms: messenger-activated target affinity (MATA) and target-activated messenger affinity (TAMA). The C2 domains studied here both utilize the TAMA mechanism, in which the C2 domain Ca 2+ affinity is too low to be activated by physiological Ca 2+ signals in most regions of the cell. Only when the C2 domain nears its target membrane, which provides a high local concentration of target lipid, is the effective Ca 2+ affinity increased by the coupled binding equilibrium to a level that enables substantial Ca 2+ activation and target docking. Overall, the findings emphasize the importance of using physiological ligand concentrations in targeting studies because super-physiological concentrations can drive docking interactions even when an important targeting molecule is missing.Many signaling pathways are regulated by signaling lipids, membrane proteins, or membranebound complexes associated with the plasma or internal cell membranes. Such membraneassociated signaling components control essential processes, such as cellular movement, growth, gene regulation, metabolism, hormone release, and inflammation. One of the most common regulatory elements in membrane-associated signaling pathways is the C2 domain, a ubiquitous, conserved signaling motif recognized in over 200 mammalian proteins (1). Structurally, the C2 domain motif comprises eight antiparallel β-strands assembled in a β- † F...

Protein kinase C isoform alpha (PKCα) is a ubiquitous, conventional PKC enzyme that possesses a conserved C2 domain. Upon activation by cytoplasmic Ca2+ ions, the C2 domain specifically binds to the plasma membrane inner leaflet where it recognizes the target lipids phosphatidylserine (PS) and phosphatidylinositol-4,5-bisphosphate (PIP2). The membrane penetration depth and docking angle of the membrane-associated C2 domain is not well understood. The present study employs EPR site-directed spin labeling and relaxation methods to generate a medium-resolution model of the PKCα C2 domain docked to a membrane of lipid composition similar to the plasma membrane inner leaflet. The approach measures EPR depth parameters for 10 function-retaining spin labels coupled to the C2 domain, and for spin labels coupled to depth calibration molecules. The resulting depth parameters, together with the known structure of the free C2 domain, provide a sufficient number of constraints to define two membrane docking geometries for C2 domain bound to physiological membranes lacking or containing PIP2, respectively. In both the absence and presence of PIP2, the two bound Ca2+ ions of the C2 domain lie near the anionic phosphate plane in the headgroup region, consistent with the known ability of the Ca2+ and membrane-binding loops (CMBLs) to bind the headgroup of the PS target lipid. In the absence of PIP2, the polybasic lipid binding site on the β3-β4 hairpin is occupied with PS, but in the presence of PIP2 this larger, higher affinity target lipid competitively displaces PS and causes the long axis of the domain to tilt 40 ± 10° toward the bilayer normal. The ability of the β3-β4 hairpin site to bind PS as well as PIP2 extends the lifetime of the membrane-docked state and is predicted to enhance the kinase turnover number of PKCα during a single membrane docking event. In principle, PIP2-induced tilting of the C2 domain could modulate the activity of membrane-docked PKCα as it diffuses between membrane regions with different local PS and PIP2 concentrations. Finally, the results demonstrate that EPR relaxation methods are sufficiently sensitive to detect signaling-induced changes in the membrane docking geometries of peripheral membrane proteins.

Protein kinase Ca (PKCα) possesses a conserved C2 domain (PKCα C2) that acts as a Ca2+-regulated membrane targeting element. Upon activation by Ca2+, PKCα C2 directs the kinase protein to the plasma membrane, thereby stimulating an array of cellular pathways. At sufficiently high Ca2+ concentrations the binding of the C2 domain to the target lipid phosphatidylserine (PS) is sufficient to drive membrane association, but at typical physiological Ca2+ concentrations binding both to PS and to phosphoinositidyl-4,5-bisphosphate (PIP2) is required for specific plasma membrane targeting. Recent EPR studies have revealed the membrane docking geometries of PKCα C2 docked to (i) PS alone, and to (ii) both PS and PIP2 simultaneously. These two EPR docking geometries exhibit significantly different tilt angles relative to the plane of the membrane, presumably induced by the large size of the PIP2 headgroup. The present study utilizes the two EPR docking geometries as starting points for molecular dynamics simulations that investigate the atomic features of the protein-membrane interaction. The simulations yield approximately the same PIP2-triggered change in tilt angle observed by EPR. Moreover, the simulations predict a PIP2:C2 stoichiometry approaching 2:1 at high PIP2 mole density. Direct binding measurements titrating the C2 domain with PIP2 in lipid bilayers yield a 1:1 stoichiometry at moderate mole densities, and a saturating 2:1 stoichiometry at high PIP2 mole densities. Thus, experiment confirms the target lipid stoichiometry predicted by EPR-guided molecular dynamics simulations. Potential biological implications of the observed docking geometries and PIP2 stoichiometries are discussed.