Macromolecular Signaling Complexes Research Articles

Mechanism of Analgesia by Gabapentinoid Drugs: Involvement of Modulation of Synaptogenesis and Trafficking of Glutamate-Gated Ion Channels.

Gabapentinoids have clinically been used for treating epilepsy, neuropathic pain, and several other neurologic disorders for >30 years; however, the definitive molecular mechanism responsible for their therapeutic actions remained uncertain. The conventional pharmacological observation regarding their efficacy in chronic pain modulation is the weakening of glutamate release at presynaptic terminals in the spinal cord. While the α2/δ-1 subunit of voltage-gated calcium channels (VGCCs) has been identified as the primary drug receptor for gabapentinoids, the lack of consistent effect of this drug class on VGCC function is indicative of a minor role in regulating this ion channel's activity. The current review targets the efficacy and mechanism of gabapentinoids in treating chronic pain. The discovery of interaction of α2/δ-1 with thrombospondins established this protein as a major synaptogenic neuronal receptor for thrombospondins. Other findings identified α2/δ-1 as a powerful regulator of N-methyl-D-aspartate receptor (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) by potentiating the synaptic expression, a putative pathophysiological mechanism of neuropathic pain. Further, the interdependent interactions between thrombospondin and α2/δ-1 contribute to chronic pain states, while gabapentinoid ligands efficaciously reverse such pain conditions. Gabapentin normalizes and even blocks NMDAR and AMPAR synaptic targeting and activity elicited by nerve injury. SIGNIFICANCE STATEMENT: Gabapentinoid drugs are used to treat various neurological conditions including chronic pain. In chronic pain states, gene expression of cacnα2/δ-1 and thrombospondins are upregulated and promote aberrant excitatory synaptogenesis. The complex trait of protein associations that involve interdependent interactions between α2/δ-1 and thrombospondins, further, association of N-methyl-D-aspartate receptor and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor with the C-tail of α2/δ-1, constitutes a macromolecular signaling complex that forms the crucial elements for the pharmacological mode of action of gabapentinoids.

Structural and Functional Coupling of Calcium-Activated BK Channels and Calcium-Permeable Channels Within Nanodomain Signaling Complexes.

Biochemical and functional studies of ion channels have shown that many of these integral membrane proteins form macromolecular signaling complexes by physically associating with many other proteins. These macromolecular signaling complexes ensure specificity and proper rates of signal transduction. The large-conductance, Ca2+-activated K+ (BK) channel is dually activated by membrane depolarization and increases in intracellular free Ca2+ ([Ca2+]i). The activation of BK channels results in a large K+ efflux and, consequently, rapid membrane repolarization and closing of the voltage-dependent Ca2+-permeable channels to limit further increases in [Ca2+]i. Therefore, BK channel-mediated K+ signaling is a negative feedback regulator of both membrane potential and [Ca2+]i and plays important roles in many physiological processes and diseases. However, the BK channel formed by the pore-forming and voltage- and Ca2+-sensing α subunit alone requires high [Ca2+]i levels for channel activation under physiological voltage conditions. Thus, most native BK channels are believed to co-localize with Ca2+-permeable channels within nanodomains (a few tens of nanometers in distance) to detect high levels of [Ca2+]i around the open pores of Ca2+-permeable channels. Over the last two decades, advancement in research on the BK channel’s coupling with Ca2+-permeable channels including recent reports involving NMDA receptors demonstrate exemplary models of nanodomain structural and functional coupling among ion channels for efficient signal transduction and negative feedback regulation. We hereby review our current understanding regarding the structural and functional coupling of BK channels with different Ca2+-permeable channels.

Role for Fgr and Numb in retinoic acid-induced differentiation and G0 arrest of non-APL AML cells.

Retinoic acid (RA) is a fundamental regulator of cell cycle and cell differentiation. Using a leukemic patient-derived in vitro model of a non-APL AML, we previously found that RA evokes activation of a macromolecular signaling complex, a signalosome, built of numerous MAPK-pathway-related signaling molecules; and this signaling enabled Retinoic-Acid-Response-Elements (RAREs) to regulate gene expression that results in cell differentiation/cell cycle arrest. Toward mechanistic insight into the nature of this novel signaling, we now find that the NUMB cell fate determinant protein is an apparent scaffold for the signalosome. Numb exists in the cell bound to an ensemble of signalosome molecules, including Raf, Lyn, Slp-76, and Vav. Addition of RA induces the expression of Fgr. Fgr binds NUMB, which is associated with (p-tyr)phosphorylation of NUMB and enhanced NUMB-binding and (p-tyr)phosphorylation of select signalosome components, thereby betraying signalosome activation. Signalosome activation is associated with cell differentiation along the myeloid lineage and G1/0 cell cycle arrest. If RA-induced Fgr expression is ablated by a CRISPR-KO; then the RA-induced (p-tyr) phosphorylation of NUMB and enhanced NUMB-binding and (p-tyr)phosphorylation of select signalosome components are lost. The cells now fail to undergo RA-induced differentiation or G1/0 arrest. In sum we find that NUMB acts as a scaffold for a signaling machine that functions to propel RA-induced differentiation and G1/0 arrest, and that Fgr binding to NUMB turns the function on. The Numb fate determinant protein thus appears to regulate the retinoic acid embryonic morphogen using the Fgr Src-Family-Kinase. These mechanistic insights suggest therapeutic targets for a hitherto incurable AML.

Caspases in Cell Death, Inflammation, and Pyroptosis.

Caspases are a family of conserved cysteine proteases that play key roles in programmed cell death and inflammation. In multicellular organisms, caspases are activated via macromolecular signaling complexes that bring inactive procaspases together and promote their proximity-induced autoactivation and proteolytic processing. Activation of caspases ultimately results in programmed execution of cell death, and the nature of this cell death is determined by the specific caspases involved. Pioneering new research has unraveled distinct roles and cross talk of caspases in the regulation of programmed cell death, inflammation, and innate immune responses. In-depth understanding of these mechanisms is essential to foster the development of precise therapeutic targets to treat autoinflammatory disorders, infectious diseases, and cancer. This review focuses on mechanisms governing caspase activation and programmed cell death with special emphasis on the recent progress in caspase cross talk and caspase-driven gasdermin D-induced pyroptosis.

CG-NAP/Kinase Interactions Fine-Tune T Cell Functions.

CG-NAP, also known as AKAP450, is an anchoring/adaptor protein that streamlines signal transduction in various cell types by localizing signaling proteins and enzymes with their substrates. Great efforts are being devoted to elucidating functional roles of this protein and associated macromolecular signaling complex. Increasing understanding of pathways involved in regulating T lymphocytes suggests that CG-NAP can facilitate dynamic interactions between kinases and their substrates and thus fine-tune T cell motility and effector functions. As a result, new binding partners of CG-NAP are continually being uncovered. Here, we review recent advances in CG-NAP research, focusing on its interactions with kinases in T cells with an emphasis on the possible role of this anchoring protein as a target for therapeutic intervention in immune-mediated diseases.

From atoms to physiology: what it takes to really understand inflammasomes.

Rapid inflammatory responses to cytosolic threats are mediated by inflammasomes - large macromolecular signalling complexes that control the activation of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18, as well as cell death by pyroptosis. Different inflammasome sensors are activated by diverse direct and indirect signals, and subsequently nucleate the polymerization of the adaptor molecule ASC to form signalling platforms macroscopically observed as ASC specks. Caspase-1 is autocatalytically activated at these sites and subsequently matures pro-inflammatory cytokines and the pore-forming effector molecule gasdermin D. While most molecules and basic assembly principles have been deduced from reductionist experimental systems, we still lack fundamental information on the structure and regulation of these complexes in their physiological environment and in the interplay with other signalling pathways. In this review, novel experimental approaches are proposed, including some that rely on nanobodies and single domain antibodies, to understand inflammasome assembly and regulation in the context of the relevant tissues or cells.

Phosphorylation-dependent modulation of CFTR macromolecular signalling complex activity by cigarette smoke condensate in airway epithelia

Genetic and acquired loss-of-function defect of the cystic fibrosis transmembrane conductance regulator (CFTR) compromise airway surface liquid homeostasis and mucociliary clearance (MCC), culminating in recurrent lung inflammation/infection. While chronic cigarette smoke (CS), CS extract (CSE; water-soluble compounds) and CS condensate (CSC; particulate, organic fraction) exposure inhibit CFTR activity at transcriptional, biochemical, and functional levels, the acute impact of CSC remains incompletely understood. We report that CSC transiently activates CFTR chloride secretion in airway epithelia. The comparable CFTR phospho-occupancy after CSC- and forskolin-exposure, determined by affinity-enriched tandem mass spectrometry and pharmacology, suggest that localised cAMP-dependent protein kinase (PKA) stimulation by CSC causes the channel opening. Due to the inhibition of the MRP4/ABCC4, a cAMP-exporter confined to the CFTR macromolecular signalling-complex, PKA activation is accomplished by the subcompartmentalised elevation of cytosolic cAMP. In line, MRP4 inhibition results in CFTR activation and phospho-occupancy similar to that by forskolin. In contrast, acute CSC exposure reversibly inhibits the phosphorylated CFTR both in vivo and in phospholipid bilayers, without altering its cell surface density and phospho-occupancy. We propose that components of CSC elicit both a transient protective CFTR activation, as well as subsequent channel block in airway epithelia, contributing to the subacute MCC defect in acquired CF lung diseases.

Molecular Mechanisms for the Mechanical Modulation of Airway Responsiveness.

The smooth muscle of the airways is exposed to continuously changing mechanical forces during normal breathing. The mechanical oscillations that occur during breathing have profound effects on airway tone and airway responsiveness both in experimental animals and humans in vivo and in isolated airway tissues in vitro. Experimental evidence suggests that alterations in the contractile and mechanical properties of airway smooth muscle tissues caused by mechanical perturbations result from adaptive changes in the organization of the cytoskeletal architecture of the smooth muscle cell. The cytoskeleton is a dynamic structure that undergoes rapid reorganization in response to external mechanical and pharmacologic stimuli. Contractile stimulation initiates the assembly of cytoskeletal/extracellular matrix adhesion complex proteins into large macromolecular signaling complexes (adhesomes) that undergo activation to mediate the polymerization and reorganization of a submembranous network of actin filaments at the cortex of the cell. Cortical actin polymerization is catalyzed by Neuronal-Wiskott–Aldrich syndrome protein (N-WASP) and the Arp2/3 complex, which are activated by pathways regulated by paxillin and the small GTPase, cdc42. These processes create a strong and rigid cytoskeletal framework that may serve to strengthen the membrane for the transmission of force generated by the contractile apparatus to the extracellular matrix, and to enable the adaptation of smooth muscle cells to mechanical stresses. This model for the regulation of airway smooth muscle function can provide novel perspectives to explain the normal physiologic behavior of the airways and pathophysiologic properties of the airways in asthma.

Applications for Mass Spectrometry in theStudy of Ion Channel Structure and Function.

Ion channels are intrinsic membrane proteins that form gated ion permeable pores across biological membranes. Depending on the type, ion channels exhibit sensitivities to a diverse range of stimuli including changes in membrane potential, binding by diffusible ligands, changes in temperature and direct mechanical force. The purpose of these proteins is to facilitate the passive diffusion of ions down their respective electrochemical gradients into and out of the cell, and between intracellular compartments. In doing so, ion channels can affect transmembrane potentials and regulate the intracellular homeostasis of the important second messenger, Ca2+, modulating a multitude of cell signaling systems in the process. The ion channels of the plasma membrane are of particular clinical interest due to their regulation of cell excitability and cytosolic Ca2+ levels, and the fact that they are particularly amenable to manipulation by exogenously applied drugs and toxins. A critical step in improving the pharmacopeia of chemicals available that influence the activity of ion channels is understanding how their three-dimensional structure relates to their function. Historically, elucidation of the structure of membrane proteins has been slow relative to that for soluble proteins, due to limitations inherent in the most widely used methods, in particular X-ray crystallography. Over the course of the last decade, starting with significant advances in X-ray crystallography followed by the more recent, and profound, surge in the use of single particle cryo-electron microscopy (cryo-EM), a slew of high resolution ion channel structures have been resolved. Overshadowed during this period have been the equally marked advances in mass spectrometry, pushing this method to the fore as an important complimentary approach to studying the structure and function of ion channels. In addition to revealing the subtle conformational changes in ion channel structure that accompany gating and permeation, mass spectrometry is already being used effectively for identifying tissue-specific posttranslational modifications and mRNA splice variants. Furthermore, the use of mass spectrometry for high throughput proteomics analysis, which has proven so successful for soluble proteins, is already providing valuable insight into the functional interactions of ion channels within the context of the macromolecular signaling complexes that they inhabit in vivo. In this chapter, the potential for mass spectrometry as a complementary approach to the study of ion channel structure and function will be reviewed with examples of its application.

Functional Microdomains in Heart's Pacemaker: A Step Beyond Classical Electrophysiology and Remodeling.

Spontaneous beating of the sinoatrial node (SAN), the primary pacemaker of the heart, is initiated, sustained, and regulated by a complex system that integrates ion channels and transporters on the cell membrane surface (often referred to as “membrane clock”) with subcellular calcium handling machinery (by parity of reasoning referred to as an intracellular “Ca2+ clock”). Stable, rhythmic beating of the SAN is ensured by a rigorous synchronization between these two clocks highlighted in the coupled-clock system concept of SAN timekeeping. The emerging results demonstrate that such synchronization of the complex pacemaking machinery at the cellular level depends on tightly regulated spatiotemporal signals which are restricted to precise sub-cellular microdomains and associated with discrete clusters of different ion channels, transporters, and regulatory receptors. It has recently become evident that within the microdomains, various proteins form an interacting network and work together as a part of a macromolecular signaling complex. These protein–protein interactions are tightly controlled and regulated by a variety of neurohormonal signaling pathways and the diversity of cellular responses achieved with a limited pool of second messengers is made possible through the organization of essential signal components in particular microdomains. In this review, we highlight the emerging understanding of the functionality of distinct subcellular microdomains in SAN myocytes and their functional role in the accumulation and neurohormonal regulation of proteins involved in cardiac pacemaking. We also demonstrate how changes in scaffolding proteins may lead to microdomain-targeted remodeling and regulation of pacemaker proteins contributing to SAN dysfunction.

Ezrin-anchored PKA phosphorylates serine 369 and 373 on connexin 43 to enhance gap junction assembly, communication, and cell fusion.

A limited number of human cells can fuse to form multinucleated syncytia. In the differentiation of human placenta, mononuclear cytotrophoblasts fuse to form an endocrinologically active, non-proliferative, multinucleated syncytium. This syncytium covers the placenta and manages the exchange of nutrients and gases between maternal and fetal circulation. We recently reported protein kinase A (PKA) to be part of a macromolecular signaling complex with ezrin and gap junction protein connexin 43 (Cx43) that provides cAMP-mediated control of gap junction communication. Here, we examined the associated phosphorylation events. Inhibition of PKA activity resulted in decreased Cx43 phosphorylation, which was associated with reduced trophoblast fusion and differentiation. In vitro studies using peptide arrays, together with mass spectrometry, pointed to serine 369 and 373 of Cx43 as the major PKA phosphorylation sites that increases gap junction assembly at the plasmalemma. A combination of knockdown and reconstitution experiments and gap-fluorescence loss in photobleaching assays with mutant Cx43 containing single or double phosphoserine-mimicking amino acid substitutions in putative PKA phosphorylation sites demonstrated that phosphorylation of S369 and S373 mediated gap junction communication, trophoblast differentiation, and cell fusion.

Differentially Expressed MicroRNAs in Conservatively Treated Nontraumatic Osteonecrosis Compared with Healthy Controls.

Deregulation of microRNAs (miRNAs) contributes to nontraumatic osteonecrosis of the femoral head (ONFH-N), but the differentially expressed circulating miRNAs in patients with ONFH-N receiving nonsurgical therapy are unknown. This study aimed to determine the miRNAs expression profile of patients with ONFH-N receiving conservative treatments. This was a case-control prospective study of 43 patients with ONFH-N and 43 participants without ONFH-N, enrolled from 10/2014 to 10/2016 at the Department of Orthopedics of the Linyi People's Hospital (China). The two groups were matched for age, gender, and living area. Microarray analysis and quantitative RT-PCR were used to examine the differentially expressed miRNAs. Bioinformatics was used to predict miRNA target genes and signaling pathways. Microarray and quantitative RT-PCR revealed that nine miRNAs were downregulated and five miRNAs were upregulated in ONFH-N (n = 3) compared with controls (n = 3). Bioinformatics showed that calcium-mediated signaling pathway, regulation of calcium ion transmembrane transporter activity, cytoskeletal protein binding, and caveolae macromolecular signaling complex were probably regulated by the identified differentially expressed miRNAs. In the remaining 80 subjects (n = 40/group), miR-335-5p was downregulated (P = 0.01) and miR-100-5p was upregulated (P = 0.02) in ONFH-N compared with controls. In conclusion, some miRNAs are differentially expressed in conservatively treated ONFH-N compared with controls. Those miRNAs could contribute to the pathogenesis of ONFH-N.

Abstract 2313: Promoting caspase-8-dependent apoptosis signaling using 17-beta-hydroxywithanolides

Abstract We have previously reported that withanolide E (WE), a steroidal lactone from Physalis peruviana, was highly active in sensitizing various human carcinoma cell lines to TRAIL-mediated apoptosis. Therefore, over 100 natural and semi-synthetic withanolides were evaluated for their ability to promote caspase-8-dependent cancer cell death. Our studies identified several withanolides that were 4-8 fold more potent than WE in sensitizing the renal carcinoma cells and melanoma cells to caspase-8-dependent apoptosis in response to either TRAIL or the TLR3 ligand poly (I:C). All active withanolides were 17-beta-hydroxywithanolides (17-BHW). The highly active 17-BHWs were more efficient than withanolide E at reducing cellular levels of both cFLIPL and cFLIPS and enhancing caspase-8 activation. Furthermore, immunoprecipitation of the TRAIL death-inducing signaling complex (DISC), or the related ripoptosome, demonstrated enhanced levels of both FADD and RIP1 in these macromolecular apoptosis signaling complexes following treatment with active 17-BHWs. The 17-BHWs used in this work were obtained by the application of an efficient method of plant biomass production involving our innovative and patented soil-less aeroponic cultivation of P. crassifolia and P. peruviana and by chemical modification of natural withanolides produced by these plants. Preliminary structure activity relationship (SAR) studies suggested that the enone moiety in ring A was essential for activity. In addition, acetoxylation at C-18, an alpha orientation of the side-chain lactone group and the double bond at C-24(25) of the lactone ring played important roles in determining the activity of 17-BHWs as apoptosis sensitizers. This suggests that the 17-BHW scaffold is amenable to optimization by a medicinal chemistry approach, which could lead to the identification of highly active natural product-based sensitizers of cancer cells to caspase-8-dependent apoptosis. The cellular molecular target(s) of active 17-BHWs are currently under further investigation. Funded by FNLCR Contract HHSN261200800001E. Citation Format: Alan D. Brooks, Ya-ming Xu, E. M. Kithsiri Wijeratne, Curtis J. Henrich, Poonam Tewary, Leslie Gunatilaka, Thomas J. Sayers. Promoting caspase-8-dependent apoptosis signaling using 17-beta-hydroxywithanolides [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2313. doi:10.1158/1538-7445.AM2017-2313

Abstract 2159: A specific 17-beta-hydroxywithanolide (LG-02) sensitizes cancer cells to apoptosis in response to TRAIL and TLR3 ligands

Abstract Recent studies have demonstrated a role of toll-like receptor 3 (TLR3) signaling for the initiation of apoptosis in some malignant cells. We have previously shown that, withanolide E (WE), a 17β-hydroxywithanolide (17-BHW) natural product derived from the medicinal plant Physalis peruviana was capable of sensitizing tumor cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis by reducing cellular levels of the anti-apoptotic protein cFLIP. Animal studies also revealed that WE sensitized human renal carcinoma cells to apoptosis at concentrations that did not promote apoptosis in normal cells. Thus we further screened a library of 30 natural and semi-synthetic 17-BHWs for their ability to promote death ligand-mediated cancer cell death. Among the 30 compounds tested, LG-02 (physachenolide C) was found to be 4-5 fold more potent than WE in sensitizing some human renal carcinoma and melanoma cells to apoptotic cell death in response not only to TRAIL but also to the synthetic polynucleotide poly (I:C), which is known to mimic anti-viral responses by activating TLR3 signaling. To date there are no withanolides known to have this dual apoptosis sensitizing activity. LG-02 and Poly (I:C) treatment resulted in increased activation of caspase-8, and apoptosis was blocked by the pan caspase inhibitor zVAD-FMK. Poly (I:C)-driven apoptosis signaling was dependent on endosomal acidification, but independent of IRF 3 and Interferon α/β signaling. Molecular studies suggested a role for changes in the anti-apoptotic proteins cFLIP, IAPs, and Livin on apoptosis signaling in LG-02 treated cells. Loss of cIAP activity is reported to promote spontaneous formation of an intracellular death-inducing protein platform the ripoptosome, that can activate either apoptosis or necroptosis. Immunoprecipitation of either the TRAIL death-inducing signaling complex (DISC) or the Poly (I:C) ripoptosome, demonstrated enhanced levels of FADD and RIP1 and decreased levels of cFLIP in these macromolecular apoptosis signaling complexes in LG-02 treated cells. Intratumor administration of LG-02 and Poly (I:C) in a xenograft M14 melanoma model provided therapeutic benefit leading to complete tumor regression in 90 % of the mice as compared to control mice. Further studies with active 17-BHWs could lead to the identification of more potent analogues, and novel and common therapeutic targets involved in apoptosis signaling in response to both TNF death receptor family members as well as TLR3 ligands.Funded by FNLCR Contract HHSN261200800001E Citation Format: Poonam Tewary, Alan D. Brooks, Ya-ming Xu, Kithsiri E.M Wijeratne, Leslie A. Gunatilaka, Thomas J. Sayers. A specific 17-beta-hydroxywithanolide (LG-02) sensitizes cancer cells to apoptosis in response to TRAIL and TLR3 ligands [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2159. doi:10.1158/1538-7445.AM2017-2159

Boosting the signal: Endothelial inward rectifier K+ channels.

Endothelial cells express a diverse array of ion channels including members of the strong inward rectifier family composed of KIR 2 subunits. These two-membrane spanning domain channels are modulated by their lipid environment, and exist in macromolecular signaling complexes with receptors, protein kinases and other ion channels. Inward rectifier K+ channel (KIR ) currents display a region of negative slope conductance at membrane potentials positive to the K+ equilibrium potential that allows outward current through the channels to be activated by membrane hyperpolarization, permitting KIR to amplify hyperpolarization induced by other K+ channels and ion transporters. Increases in extracellular K+ concentration activate KIR allowing them to sense extracellular K+ concentration and transduce this change into membrane hyperpolarization. These properties position KIR to participate in the mechanism of action of hyperpolarizing vasodilators and contribute to cell-cell conduction of hyperpolarization along the wall of microvessels. The expression of KIR in capillaries in electrically active tissues may allow KIR to sense extracellular K+ , contributing to functional hyperemia. Understanding the regulation of expression and function of microvascular endothelial KIR will improve our understanding of the control of blood flow in the microcirculation in health and disease and may provide new targets for the development of therapeutics in the future.

Adenylyl Cyclase Isoform Specific Effects in Lipid Raft and Non‐Lipid Raft Membrane Domains Regulate cAMP Compartmentation in Human Airway Smooth Muscle Cells

The formation of distinct macromolecular signaling complexes allows different G‐protein coupled receptors to produce diverse functional responses, even while sharing a common second messenger such as cAMP. In human airway smooth muscle (HASM) cells, segregation of specific receptors into different membrane microdomains is thought to critically aid in generating compartmentalized cAMP responses. Whereas, E type prostaglandin receptors (EPRs) have been shown to be expressed in non‐lipid raft domains of the plasma membrane, β‐Adrenergic receptors (βARs) are predominantly expressed in caveolar lipid rafts. In the present study, we tested the hypothesis that adenylyl cyclase type 2 (AC2) preferentially couples to EPRs in a non‐lipid raft domain, while adenylyl cyclase type 6 (AC6) selectively couples to βARs in lipid rafts. To do this, we examined the effect of overexpressing AC2 and AC6 on cAMP responses detected using genetically‐encoded FRET‐based biosensors targeted to lipid raft and non‐lipid raft domains of the plasma membrane, as well as the bulk cytosolic compartment in HASM cells. This approach has the advantage of measuring the kinetics of cAMP production in living cells without the use of PDE inhibitors. Overexpression of AC2 enhanced the cAMP response to EPR activation associated with non‐lipid raft domains, without significantly affecting responses detected elsewhere. AC2 overexpression also had no effect on cAMP responses to βAR activation detected in any subcellular location. These data confirm the hypothesis that AC2 couples exclusively with EPRs in a non‐lipid raft membrane compartment. Overexpression of AC6, on the other hand, actually decreased the response to βAR stimulation associated with lipid rafts, without significantly affecting responses elsewhere. AC6 overexpression also had no effect on the responses to EPR activation detected anywhere in the cell. The ability of AC6 overexpression to inhibit βAR production of cAMP in lipid raft domains was reversed by inhibition of PDE4 activity with rolipram. It was also reversed by overexpression of Ht31 peptide, which disrupts the interaction of protein kinase A with A‐kinase anchoring proteins (AKAPs). These results suggests that AC6 overexpression upregulates and/or recruits PKA and PDE4 activity, which then reduces βAR production of cAMP associated specifically with lipid raft domains.Support or Funding InformationSupport by: NIH R01 GM107094

Ankyrin-rich membrane spanning protein as a novel modulator of transient receptor potential vanilloid 1-function in nociceptive neurons.

The ion channel TRPV1 is mainly expressed in small diameter dorsal root ganglion (DRG) neurons, which are involved in the sensation of acute noxious thermal and chemical stimuli. Direct modifications of the channel by diverse signalling events have been intensively investigated, but little is known about the composition of modulating macromolecular TRPV1 signalling complexes. Here, we hypothesize that the novel adaptor protein ankyrin-rich membrane spanning protein/kinase D interacting substrate (ARMS) interacts with TRPV1 and modulates its function in rodent DRG neurons. We used immunohistochemistry, electrophysiology, microfluorimetry and immunoprecipitation experiments to investigate TRPV1 and ARMS interactions in DRG neurons and transfected cells. We found that TRPV1 and ARMS are co-expressed in a subpopulation of DRG neurons. ARMS sensitizes TRPV1 towards capsaicin in transfected HEK 293 cells and in mouse DRG neurons in a PKA-dependent manner. Using a combination of functional imaging and immunocytochemistry, we show that the magnitude of the capsaicin response in DRG neurons depends not only on TRPV1 expression, but on the co-expression of ARMS alongside TRPV1. These data indicate that ARMS is an important component of the signalling complex regulating the sensitivity of TRPV1. The study identifies ARMS as an important component of the signalling complex regulating the sensitivity of excitatory ion channels (TRPV1) in peripheral sensory neurons (DRG neurons) and transfected cells.

Loss of Caveolin-3 Causes Heart Rhythm Abnormalities by Affecting Ca2+-Voltage Coupling in the Mouse Sinoatrial Node

Caveolin-3 (Cav3) is a myocyte-specific scaffolding protein within caveolar membranes that compartmentalize a number of ion channels and transporters involved in sinoatrial node (SAN) pacemaking. These channels and transporters are coupled with intracellular Ca2+ cycling (Ca2+-voltage coupling) through rhythmic local Ca2+ releases (LCRs) from ryanodine receptors to initiate an action potential. We hypothesize that disruption of caveolae-associated macromolecular signaling complexes alters SAN automaticity leading to SAN dysfunction. In vivo ECG telemetry, in vitro high-resolution optical mapping, and confocal imaging of intracellular Ca2+ cycling were performed in wild type (WT, n=8) and conditional tamoxifen-induced α-MHC-controlled Cav3 knockout (Cav3-/-, n=8) mice and isolated SAN myocytes. Both in vivo and in vitro,Cav3-/- mice exhibited SAN pacemaking abnormalities characterized by alternating periods of tachycardia-bradycardia rhythm (cycle length varied from 123.6±2.6 ms to 464.4±130 ms), significant beat-to-beat cycle length variability (3.8±1.1 ms in WT vs. 19.5±3.6 ms in Cav3-/-, p<0.01) and shift of the leading pacemaker from the SAN to ectopic foci. In SAN myocytes, Cav3 deficiency led to irregular spontaneous rate with significant cycle length variability (30.9±5.2 ms in WT vs. 644.7±256.4 ms in Cav3-/-, p<0.01). Because LCRs are critically important for the Na+/Ca2+ exchanger mediated component of slow diastolic depolarization of transmembrane potential, we measured the period from LCRs to Ca2+ transient initiation in order to evaluate Ca2+-voltage coupling. Cav3-/- SAN myocytes showed significant prolongation (73.9±6.7 ms in WT vs. 130.6±15.0 ms in Cav3-/-, p<0.01) of this period with large beat-to-beat variability (15.5±2.6 ms in WT vs. 71.6±17.4 ms in Cav3-/-, p<0.01). Our findings demonstrate that Cav3 plays a crucial role in supporting functional integrity of the SAN by synchronizing Ca2+-voltage coupling and regulating rhythm of LCRs.

PKA: Dynamic Assembly and Regulation of Macromolecular Signaling Complexes

PKA Signaling: Linkers, Loops, and Intrinsically Disorder Regions Drive the Dynamic Assembly of Holoenzyme Complexes.The PKA catalytic (C) subunit crystallized in 1991, gave us our first glimpse of the fold that describes all protein kinases. The two lobes that comprise the core are flanked by N-and C-terminal tails, which wrap around both lobes. By capturing all stages of catalysis in crystal structures we appreciate the dynamic nature of these tails and how they undergo order-disorder transitions as part of the catalytic cycle. Ordering the activation loop by a critical phosphate is another fundamental order/disorder transition that is a conserved feature of most protein kinases. These dynamic features allow the kinases to function as highly regulated molecular switches. In the case of PKA, the C-subunit is packaged with regulatory (R) subunit dimers where each chain contains two C-terminal cAMP binding (CNB) domains. The CNB domains are highly dynamic allosteric signaling modules that have been conserved to translate a biological response to cAMP. Although the fundamental features of this domain were elucidated by structures of R-monomers and R:C heterodimers, it required full-length R2C2 tetramers to appreciate the importance of symmetry for PKA signaling. Each R-subunit contains an N-terminal dimerization domain that is also a docking site for scaffold proteins referred to as A Kinase Anchoring Proteins (AKAPs). This domain is joined by a flexible linker to the CNB domains, and embedded within the linker is an inhibitor site that docks to the active site of the C-subunit in the holoenzyme. The order/disorder transitions of the linker drive the assembly of the holoenzymes which each have distinct quaternary structures. The linkers, rich in biological information, are an essential feature of PKA regulation. (Funded by NIH GM34921 and DK54441.)

Force Matters: Biomechanical Regulation of Cell Invasion and Migration in Disease.

Atherosclerosis, cancer, and various chronic fibrotic conditions are characterized by an increase in the migratory behavior of resident cells and the enhanced invasion of assorted exogenous cells across a stiffened extracellular matrix (ECM). This stiffened scaffold aberrantly engages cellular mechanosignaling networks in cells, which promotes the assembly of invadosomes and lamellae for cell invasion and migration. Accordingly, deciphering the conserved molecular mechanisms whereby matrix stiffness fosters invadosome and lamella formation could identify therapeutic targets to treat fibrotic conditions, and reducing ECM stiffness could ameliorate disease progression.