Entire Cytoplasmic Domain Research Articles

Subunit-specific inhibition of BK channels by piperine

Piperine is the principal alkaloid present in black pepper and is well-known for its diverse pharmacological effects, including inhibition of different ion channels. Large conductance Ca2+-activated K+ channels (BK) are widely expressed across several tissues and play a vital role in many physiological functions. In this study, we investigated the pharmacological effects of piperine on various BK channel subunit compositions (BKα, BKαβ1,4, BKαγ1,3) expressed in HEK293T cells. Piperine in zero Ca2+ reversibly inhibited currents from the pore-forming BKα channels in a dose-dependent manner with a half-maximal inhibitory concentration (IC50) of 4.8 μM. Elevating the internal Ca2+ concentration from 0 to 100 μM significantly attenuated the inhibitory effects of piperine on BKα channels. The mutation G311S in the pore domain failed to alter the modulatory effects of piperine, whereas deletion of the entire cytoplasmic domain from BKα channels ablated its inhibitory effects. Addition of either BKβ1 or β4 regulatory subunits did not alter the efficacy of piperine on BKα channels. Interestingly, co-expression of either BKγ1 or BKγ3 subunits greatly diminished the ability of piperine to inhibit BKα channels. Our findings demonstrate that piperine is a potent natural modulator of BKα/BKαβ1,4 subunits but not BKαγ1,3 subunits. The mechanism of piperine modulation appeared to be allosteric and differs from that of other BK pore blockers (paxilline, peptide toxins, and quaternary ammonium compounds). Together, our results unravel the potential of piperine to inhibit BK channels, providing a new tool to explore mechanisms underlying the effects of regulatory subunits.

3183 – THE ROLE OF THE THROMBOPOIETIN RECEPTOR (MPL) IN JAK2V617F-POSITIVE MYELOPLROLIFERATIVE NEOPLASMS

Constitutive activation of the Jak/Stat signalling pathway is a hallmark of Myeloproliferative Neoplasms (MPN), with 60-90% of patients carrying the activating JAK2 mutation, JAK2V617F. Several studies suggest expression of a type I cytokine receptor is essential for JAK2V617F-mediated transformation, but the precise contribution of such receptors is unresolved. Here we investigate the functional role of Mpl in JAK2V617F-driven MPN in vitro and in vivo. In vitro studies confirmed that Mpl expression was required for JAK2V617F-induced transformation, but identified a Mpl truncation mutant retaining the JAK2-interaction domain, but lacking the C-terminus (Mpl-IC36), typically associated with Stat5 binding, to be sufficient to support JAK2V617F-induced transformation. Interestingly, Stat5-phosphorylation was maintained. In contrast, deletion of the entire cytoplasmic receptor domain abrogated Stat5 phosphorylation and factor-independent proliferation. In vivo Mpl-wt;JAK2V617F mice exhibited the thrombocytosis and erythrocytosis typical of MPN, while Mpl deficiency abrogated thrombocytosis but sustained high red blood cell counts. Although the Mpl-IC36 receptor was sufficient for JAK2V617F-induced factor independence in BaF/3 cells, Mpl-IC36;JAK2V617F mice did not develop thrombocytosis, indeed platelet counts were similar to those in Mpl null mice. This underlines differences that may emerge from in vitro and in vivo systems and highlights the important role of the Mpl cytoplasmic region for JAK2V617F-mediated signalling, extending the function of Mpl in JAK2V617F-induced MPN beyond a simple scaffold for active JAK2.

PS1446 THE ROLE OF THE THROMBOPOIETIN RECEPTOR (MPL) IN JAK2V617F‐POSITIVE MYELOPLROLIFERATIVE NEOPLASMS

Background:Constitutive activation of the Jak/Stat signalling pathway is a hallmark of Myeloproliferative Neoplasms (MPN), with 60–90% of patients carrying the activating JAK2 mutation, JAK2V617F. Several studies suggest expression of a type I cytokine receptor is essential for JAK2V617F‐mediated transformation, but the precise contribution of such receptors is unresolved.Aims:Here we investigate the functional role of Mpl in JAK2V617F‐driven MPN.Methods:Mpl mutants lacking various functional domains were screened for their capacity to support JAK2V617F‐mediated factor independence in BaF/3 cells. Using CRISPR/CAS9 genome editing Mpl‐mutant mice were generated to extend these findings to an in vivo model of JAK2V617F MPN.Results:In vitro studies confirmed that Mpl expression was required for JAK2V617F‐induced transformation, but identified a Mpl truncation mutant retaining the JAK2‐interaction domain, but lacking the C‐terminus (Mpl‐IC36), typically associated with Stat5 binding, to be sufficient to support JAK2V617F‐induced transformation. Interestingly, Stat5‐phosphorylation was maintained. In contrast, deletion of the entire cytoplasmic receptor domain abrogated Stat5 phosphorylation and factor‐independent proliferation. In vivo Mpl‐wt;JAK2V617F mice exhibited the thrombocytosis and erythrocytosis typical of MPN, while Mpl deficiency abrogated thrombocytosis but sustained high red blood cell counts. Although the Mpl‐IC36 receptor was sufficient for JAK2V617F‐induced factor independence in BaF/3 cells, Mpl‐IC36;JAK2V617F mice did not develop thrombocytosis, indeed platelet counts were similar to those in Mpl null mice. This underlines differences that may emerge from in vitro and in vivo systems and highlights the important role of the Mpl cytoplasmic region for JAK2V617F‐mediated signalling.Summary/Conclusion:JAK2V617F requires Mpl to induce transformation. While a Mpl truncation mutant retaining the JAK2‐interaction domain, but lacking the C‐terminus (Mpl‐IC36) was sufficient to induce JAK2V617F‐mediated cytokine independence in BaF/3 cells in vitro, this receptor was insufficient for development of thrombocytosis in JAK2V617F mouse model. These results suggest that the role of Mpl in JAK2V617F‐induced MPN extends beyond a simple scaffold for active JAK2 and that signalling from the Mpl C‐terminal domain is required in vivo.

Transmembrane domain-mediated Lck association underlies bystander and costimulatory ICOS signaling

The B7-family inducible costimulator (ICOS) activates phosphoinositide-3 kinase (PI3K) and augments calcium mobilization triggered by the T-cell receptor (TCR). We surprisingly found that the entire cytoplasmic domain of ICOS is dispensable for its costimulation of calcium mobilization. This costimulatory function relies on the unique transmembrane domain (TMD) of ICOS, which promotes association with the tyrosine kinase Lck. TMD-enabled Lck association is also required for p85 recruitment to ICOS and subsequent PI3K activation, and Lck underlies both the bystander and costimulatory signaling activity of ICOS. TMD-replaced ICOS, even with an intact cytoplasmic domain, fails to support TFH development or GC formation in vivo. When transplanted onto a chimeric antigen receptor (CAR), the ICOS TMD enhances interactions between T cells and antigen-presenting target cells. Therefore, by revealing an unexpected function of the ICOS TMD, our study offers a new perspective for the understanding and potential application of costimulation biology.

High-affinity heterotetramer formation between the large myelin-associated glycoprotein and the dynein light chain DYNLL1.

The close association of myelinated axons and their myelin sheaths involves numerous intercellular molecular interactions. For example, myelin-associated glycoprotein (MAG) mediates myelin-to-axon adhesion and signalling via molecules on the axonal surface. However, knowledge about intracellular binding partners of myelin proteins, including MAG, has remained limited. The two splice isoforms of MAG, S- and L-MAG, display distinct cytoplasmic domains and spatiotemporal expression profiles. We used yeast two-hybrid screening to identify interaction partners of L-MAG and found the dynein light chain DYNLL1 (also termed dynein light chain 8). DYNLL1 homodimers are known to facilitate dimerization of target proteins. L-MAG and DYNLL1 associate with high affinity, as confirmed with recombinant proteins invitro. Structural analyses of the purified complex indicate that the DYNLL1-binding segment is localized close to the L-MAG C terminus, next to the Fyn kinase Tyr phosphorylation site. The crystal structure of the complex between DYNLL1 and its binding segment on L-MAG shows 2:2 binding in a parallel arrangement, indicating a heterotetrameric complex. The homology between L-MAG and previously characterized DYNLL1-ligands is limited, and some details of binding site interactions are unique for L-MAG. The structure of the complex between the entire L-MAG cytoplasmic domain and DYNLL1, as well as that of the extracellular domain of MAG, were modelled based on small-angle X-ray scattering data, allowing structural insights into L-MAG interactions on both membrane surfaces. Our data imply that DYNLL1 dimerizes L-MAG, but not S-MAG, through the formation of a specific 2:2 heterotetramer. This arrangement is likely to affect, in an isoform-specific manner, the functions of MAG in adhesion and myelin-to-axon signalling. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. Read the Editorial Highlight for this article on page712.

Intrinsic disorder in the regulatory N-terminal domain of diacylglycerol acyltransferase 1 from Brassica napus

Proteins with multifunctional regulatory domains often demonstrate structural plasticity or protein disorder, allowing the binding of multiple regulatory factors and post-translational modifications. While the importance of protein disorder is clear, it also poses a challenge for in vitro characterization. Here, we report protein intrinsic disorder in a plant molecular system, which despite its prevalence is less studied. We present a detailed biophysical characterization of the entire cytoplasmic N-terminal domain of Brassica napus diacylglycerol acyltransferase, (DGAT1), which includes an inhibitory module and allosteric binding sites. Our results demonstrate that the monomeric N-terminal domain can be stabilized for biophysical characterization and is largely intrinsically disordered in solution. This domain interacts with allosteric modulators of DGAT1, CoA and oleoyl-CoA, at micromolar concentrations. While solution scattering studies indicate conformational heterogeneity in the N-terminal domain of DGAT1, there is a small gain of secondary structure induced by ligand binding.

Intra- and extracellular domains of the Helicoverpa armigera cadherin mediate Cry1Ac cytotoxicity

Diverse midgut cadherin mutations confer resistance to Cry1A toxins in at least three lepidopteran pests, including the cotton bollworm, Helicoverpa armigera. Most of these cadherin mutations are inherited as recessive alleles and result in changes within the cadherin repeat (CR) regions of the extracellular protein domain. However, the H. armigera r15 cadherin mutation results in a deletion of 55 amino acid residues within the cytoplasmic domain, and Cry1A resistance is inherited as a non-recessive trait. Here, eight recombinant H. armigera cadherin (HaCad) proteins, including seven variants containing different combinations of CRs and the cytoplasmic domain, were expressed in cultured insect cells using a baculovirus expression system and were analyzed for Cry1Ac binding and toxicity. Cells expressing either the wild-type HaCad or a mutant lacking only the region corresponding to the first nine CRs bound Cry1Ac and were equally susceptible to Cry1Ac. Cells expressing mutant HaCad proteins without the Cry1A toxin binding region (TBR) located in the CR nearest the plasma membrane did not bind Cry1Ac and were not killed by the toxin. Among the mutant proteins, loss of toxicity was observed in all cells producing HaCad variants lacking the amino acids 1422–1440, indicating that this TBR motif is important for both toxin binding and to confer susceptibility to Cry1Ac. Cells expressing the HaCad variant lacking the entire cytoplasmic domain retained Cry1Ac binding, but were significantly less susceptible to Cry1Ac than the cells producing either wild-type HaCad or HaCad lacking the first nine CRs. These results suggest that both the extracellular and the cytoplasmic domains of HaCad participate in Cry1Ac intoxication.

Functional Analysis of the Transmembrane and Cytoplasmic Domains of Pcdh15a in Zebrafish Hair Cells

Protocadherin 15 (PCDH15) is required for mechanotransduction in sensory hair cells as a component of the tip link. Isoforms of PCDH15 differ in their cytoplasmic domains (CD1, CD2, and CD3), but share the extracellular and transmembrane (TMD) domains, as well as an intracellular domain known as the common region (CR). In heterologous expression systems, both the TMD and CR of PCDH15 have been shown to interact with members of the mechanotransduction complex. The in vivo significance of these protein-protein interaction domains of PCDH15 in hair cells has not been determined. Here, we examined the localization and function of the two isoforms of zebrafish Pcdh15a (CD1 and CD3) in pcdh15a-null mutants by assessing Pcdh15a transgene-mediated rescue of auditory/vestibular behavior and hair cell morphology and activity. We found that either isoform alone was able to rescue the Pcdh15a-null phenotype and that the CD1- or CD3-specific regions were dispensable for hair bundle integrity and labeling of hair cells with FM4-64, which was used as a proxy for mechanotransduction. When either the CR or TMD domain was deleted, the mutated proteins localized to the stereocilial tips, but were unable to rescue FM4-64 labeling. Disrupting both domains led to a complete failure of Pcdh15a to localize to the hair bundle. Our findings demonstrate that the TMD and cytoplasmic CR domains are required for the in vivo function of Pcdh15a in zebrafish hair cells.SIGNIFICANCE STATEMENT Tip links transmit force to mechanotransduction channels at the tip of hair bundles in sensory hair cells. One component of tip links is Protocadherin 15 (PCDH15). Here, we demonstrate that, when transgenically expressed, either zebrafish Pcdh15a-cytodomain 1 (CD1) or Pcdh15a-CD3 can rescue the phenotype of a pcdh15a-null mutant. Even when lacking the specific regions for CD1 or CD3, truncated Pcdh15a that contains the so-called common region (CR) at the cytoplasmic/membrane interface still has the ability to rescue similar to full-length Pcdh15a. In contrast, Pcdh15a lacking the entire cytoplasmic domain is not functional. These results demonstrate that the CR plays a key role in the mechanotransduction complex in hair cells.

Processing of heparanase is mediated by syndecan-1 cytoplasmic domain and involves syntenin and α-actinin.

Heparanase activity plays a decisive role in cell dissemination associated with cancer metastasis. Cellular uptake of heparanase is considered a pre-requisite for the delivery of latent 65-kDa heparanase to lysosomes and its subsequent proteolytic processing and activation into 8- and 50-kDa protein subunits by cathepsin L. Heparan sulfate proteoglycans, and particularly syndecan, are instrumental for heparanase uptake and activation, through a process that has been shown to occur independent of rafts. Nevertheless, the molecular mechanism underlying syndecan-mediated internalization outside of rafts is unclear. Here, we examined the role of syndecan-1 cytoplasmic domain in heparanase processing, utilizing deletion constructs lacking the entire cytoplasmic domain (Delta), the conserved (C1 or C2), or variable (V) regions. Heparanase processing was markedly increased following syndecan-1 over-expression; in contrast, heparanase was retained at the cell membrane and its processing was impaired in cells over-expressing syndecan-1 deleted for the entire cytoplasmic tail. We have next revealed that conserved domain 2 (C2) and variable (V) regions of syndecan-1 cytoplasmic tail mediate heparanase processing. Furthermore, we found that syntenin, known to interact with syndecan C2 domain, and α actinin are essential for heparanase processing.

Genetic Evidence for the Adhesion Protein IgSF9/Dasm1 to Regulate Inhibitory Synapse Development Independent of its Intracellular Domain

Normal brain function requires balanced development of excitatory and inhibitory synapses. An imbalance in synaptic transmission underlies many brain disorders such as epilepsy, schizophrenia, and autism. Compared with excitatory synapses, relatively little is known about the molecular control of inhibitory synapse development. We used a genetic approach in mice to identify the Ig superfamily member IgSF9/Dasm1 as a candidate homophilic synaptic adhesion protein that regulates inhibitory synapse development. IgSF9 is expressed in pyramidal cells and subsets of interneurons in the CA1 region of hippocampus. Electrophysiological recordings of acute hippocampal slices revealed that genetic inactivation of the IgSF9 gene resulted in fewer functional inhibitory synapses; however, the strength of the remaining synapses was unaltered. These physiological abnormalities were correlated with decreased expression of inhibitory synapse markers in IgSF9(-/-) mice, providing anatomical evidence for a reduction in inhibitory synapse numbers, whereas excitatory synapse development was normal. Surprisingly, knock-in mice expressing a mutant isoform of IgSF9 lacking the entire cytoplasmic domain (IgSF9(ΔC/ΔC) mice) had no defects in inhibitory synapse development, providing genetic evidence that IgSF9 regulates synapse development via ectodomain interactions rather than acting itself as a signaling receptor. Further, we found that IgSF9 mediated homotypic binding and cell aggregation, but failed to induce synapse formation, suggesting that IgSF9 acts as a cell adhesion molecule (CAM) to maintain synapses. Juvenile IgSF9(-/-) mice exhibited increased seizure susceptibility indicative of an imbalance in synaptic excitation and inhibition. These results provide genetic evidence for a specific role of IgSF9 in inhibitory synapse development/maintenance, presumably by its CAM-like activity.

Structural Dynamics Underlying Pip2 Modulation of Kir Channels Revealed by Single Molecule FRET

Ion channels are gated by a multitude of electrical, chemical and mechanical stimuli, and thereby play key roles in many physiological processes. Static crystal structures and dynamic electrophysiological studies have generated invaluable molecular insights into channel gating, but the time trajectory of conformational changes has been unattainable. KirBac1.1 is a model for one of the three main K channel families. In the present study, we have successfully (1) labeled purified tetrameric protein molecules with single FRET donor and acceptor fluorophore pairs at specific locations; (2) functionally reconstituted the proteins into liposomes; (3) examined the dynamics of channel structure in liposomes with single molecule FRET techniques. Our results indicate that the amphipathic 'slide helices' of KirBac1.1 that surround the channel gate immediately below the membrane, move toward each other to narrow the pore in the presence of PIP2, which closes the channel; the cytoplasmic domain resides at two major structural states, but demonstrates an overall shift away from the pore axis upon PIP2 inhibition; the extracellular loop of KirBac1.1, adjacent to the selectivity filter, is structurally rigid and does not move during PIP2 gating. Both slide helix and cytoplasmic domain structures become less dynamic when the channel is inhibited by PIP2, which implies that PIP2 acts as a 'lock' to confine the structural fluctuations of the channel protein. We further probed the relative motions of the KirBac1.1 transmembrane domain versus the cytoplasmic domain. The results indicate that the entire cytoplasmic domain moves away from the transmembrane domain during PIP2 inhibition, which may result from a rigid body motion coupled to TM2 bending. In summary, we provide direct observations of dynamic structures of an ion channel within a lipid membrane environment, revealing dynamic structural rearrangements KirBac1.1 upon PIP2 inhibition.

Platelet Cytoskeletal Force Transmission Through The GPIb-IX-V Complex

Platelets are the primary cellular components of the hemostatic plug that forms during primary hemostasis. The first step in this process is platelet adhesion from the flowing blood to a surface, carried out by the platelet glycoprotein (GP) Ib-IX-V binding to immobilized von Willebrand factor (VWF). Adhesion is followed by activation of integrin αIIbβ3, which mediates the attachment of platelets to each other by binding multivalent ligands such as VWF or fibrinogen. To stabilize the hemostatic plug and strengthen its attachment to the wound site, platelets must transmit contractile forces from actin and myosin proteins in their cytoskeleton to extracellular matrix proteins within the vessel wall or to the adhesive proteins between adjacent platelets. Integrin αIIbβ3 is one of the membrane proteins capable of transmitting these forces, having a direct link to the platelet cytoskeleton through talin and other focal adhesion related proteins. In the current study, we investigated whether the GPIb-IX-V complex is also capable of force transmission after binding ligand.The GPIb-IX-V complex contains 4 polypeptides, GPIbα, GPIbβ, GPIX and GPV. Only GPIbα binds VWF, which it does through VWF's A1 domain. GPIbα also attaches the complex to the actin and membrane skeletons through its cytoplasmic domain, with the large skeletal protein filamin functioning as the intermediary. There is strong evidence that the GPIbα-A1 bond is force sensitive, becoming stronger as force is applied to it, a property that defines it as a “catch bond”. For this reason, we investigated the role of GPIbα in transmitting platelet forces using a new tool that we have developed to measure contractile forces generated by platelets. This tool, composed of arrays of nanoposts separated by 2 μm (Figure 1), was fabricated using e-beam lithography. VWF was adsorbed to the tips of the nanoposts and platelets were allowed to adhere, spread, and contract. To assess the contribution of αIIbβ3 and GPIbα to force generation, we blocked these receptors with the antibodies 7E3 and AK2, respectively. Treatment with 7E3 significantly lowered the force generated, but did not eliminate it completely (57% reduction). AK2 had a smaller effect (20% reduction), and the combination of the two usually abolished force generation. We observed a similar force reduction (30%) as AK2 treatment when we blocked the VWF A1 domain with recombinant GPIbα N-terminus. Because VWF contains binding sites for more than one platelet receptor, and although purified, could have trace amounts of other plasma proteins, we also evaluated force generation on nanoposts coated with recombinant VWF A1 domain, which should only bind GPIbα. In this case, the platelets generated forces similar to those observed when αIIbβ3 was blocked by 7E3, providing further evidence that GPIbα can transmit forces by binding the A1 domain. [Display omitted] As a final test of the ability of GPIbα to support force generation, we examined whether Chinese hamster ovary (CHO) cells expressing the GPIb-IX complex (CHOαβIX, fully functional but lacking GPV) could generate force on VWF or A1 domain (Figure 2). CHOαβIX cells adhered, spread and generated forces of similar magnitude on microposts (larger because of the larger cell size) coated with either substrate. CHOβIX cells, lacking GPIbα, did not adhere to either substrate. [Display omitted] To investigate the requirement for cytoskeletal attachment of the complex in force generation, we studied a CHOαβIX line containing GPIbα truncated after residue 518 and therefore lacking almost the entire cytoplasmic domain. These cells adhered and spread on VWF-coated microposts, but generated minimal contractile force.Together, these results indicate that the GPIb-IX-V complex is able to transmit cytoskeletal contractile forces to its ligand, VWF, in a process requiring the cytoplasmic domain of GPIbα. This is the first example of a non-integrin transmitting force to an external substrate. Disclosures:Sniadecki:Stasys Medical Corporation: Equity Ownership, Founder Other.

Calmodulin Adopts an Extended Conformation when Interacting with L-Selectin in Membranes

Calmodulin, an intracellular calcium-binding protein, is thought to regulate ectodomain shedding of many membrane proteins, but the underlying molecular mechanism has remained unclear. Basing on a solution structure of calcium-loaded calmodulin in complex with a L-selectin fragment that contains a portion of its transmembrane domain, Gifford et al. (University of Calgary) recently suggested that calmodulin regulates L-selectin shedding by binding directly to a portion of the L-selectin transmembrane domain in a compact conformation. Using fluorescently labeled calmodulin, we show however that calmodulin adopts a distinctly different and much more extended conformation when it binds to the CLS peptide (i.e. the entire transmembrane and cytoplasmic domains of L-selectin) reconstituted in the phosphatidylcholine liposome with micromolar dissociation constant and in a calcium-independent manner. Calmodulin adopts a similarly extended conformation in a ternary complex with the N-terminal FERM domain of moesin and CLS reconstituted in the phospholipid liposome that mimics the native membrane environment. These results indicate that calmodulin does not bind directly to the transmembrane domain of L-selectin. Understanding the association of calmodulin with L-selectin helps to shed light on the mechanisms underlying regulation of ectodomain shedding.

Cell surface localization of α3β4 nicotinic acetylcholine receptors is regulated by N-cadherin homotypic binding and actomyosin contractility.

Neuronal nicotinic acetylcholine receptors (nAChRs) are widely expressed throughout the central and peripheral nervous system and are localized at synaptic and extrasynaptic sites of the cell membrane. However, the mechanisms regulating the localization of nicotinic receptors in distinct domains of the cell membrane are not well understood. N-cadherin is a cell adhesion molecule that mediates homotypic binding between apposed cell membranes and regulates the actin cytoskeleton through protein interactions with the cytoplasmic domain. At synaptic contacts, N-cadherin is commonly localized adjacent to the active zone and the postsynaptic density, suggesting that N-cadherin contributes to the assembly of the synaptic complex. To examine whether N-cadherin homotypic binding regulates the cell surface localization of nicotinic receptors, this study used heterologous expression of N-cadherin and α3β4 nAChR subunits C-terminally fused to a myc-tag epitope in Chinese hamster ovary cells. Expression levels of α3β4 nAChRs at cell-cell contacts and at contact-free cell membrane were analyzed by confocal microscopy. α3β4 nAChRs were found distributed over the entire surface of contacting cells lacking N-cadherin. In contrast, N-cadherin-mediated cell-cell contacts were devoid of α3β4 nAChRs. Cell-cell contacts mediated by N-cadherin-deleted proteins lacking the β-catenin binding region or the entire cytoplasmic domain showed control levels of α3β4 nAChRs expression. Inhibition of actin polymerization with latrunculin A and cytochalasin D did not affect α3β4 nAChRs localization within N-cadherin-mediated cell-cell contacts. However, treatment with the Rho associated kinase inhibitor Y27632 resulted in a significant increase in α3β4 nAChR levels within N-cadherin-mediated cell-cell contacts. Analysis of α3β4 nAChRs localization in polarized Caco-2 cells showed specific expression on the apical cell membrane and colocalization with apical F-actin and the actin nucleator Arp3. These results indicate that actomyosin contractility downstream of N-cadherin homotypic binding regulates the cell surface localization of α3β4 nAChRs presumably through interactions with a particular pool of F-actin.

Abstract 4544: Novel TDA technology and high content imaging as complementary assay platforms to identify VEGFR-2 inhibitors.

Abstract Vascular endothelial growth factor (VEGF) is potent mitogen for endothelial cells that signals through VEGF Receptor-type2 (VEGFR-2)/Flk-1/KDR. Binding of VEGF to VEGFR-2, a membrane bound receptor tyrosine kinase of the VEGF-receptor family, leads to receptor dimerization, protein kinase activation, trans-autophosphorylation, and initiation of signaling pathways stimulating angiogenesis. In adults, angiogenesis normally occurs during wound healing to restore blood flow post injury and during menstruation and pregnancy. Angiogenesis is also involved in several pathological disease states such as rheumatoid arthritis, psoriasis, diabetic retinopathy and cancer. While physiological angiogenesis involves interplay between pro- and anti-angiogenic factors, the ability to inhibit pathological angiogenesis has therapeutic potential. Inhibitors of VEGFR-2 provide targeted treatments for tumor growth as well as abnormal neovascularization related diseases. The caveat of traditional biochemical VEGFR-2 assays is that only the catalytic domain of the receptor is used to identify hits. One strategy to identify true novel inhibitors against membrane associated proteins is to screen these targets in a more physiological environment, where lipid-protein and protein-protein interactions are included in the assay. We achieved this goal by docking the entire cytoplasmic domain of the receptor on a liposome. Template Directed Assembly (TDA) is a screening technology designed to accomplish this objective. TDA technology utilizes recombinant VEGFR-2 cytoplasmic domain tagged with polyhistidine at the juxtamembrane position and docked to the liposome via the nickel residues. The lipids in TDA are able to diffuse within the surface of the liposome and, therefore, proteins can diffuse to form oligomeric complexes such as homo- or hetero-dimers formation. To compliment the TDA biochemical screen, we are implementing an image based high content screen (HCS) utilizing human umbilical vein endothelial cells (HUVECs) that express native VEGFR-2. The HCS screen is designed to identify potent and specific inhibitors of VEGFR-2 as assessed by a reduction in the proliferation marker Ki67. We are currently (1) validating the TDA assay with known VEGFR-2 inhibitors (2) screening a focus small molecule library to identify novel hits (3) testing the primary hits against a kinase selectivity panel and (4) designing and developing a VEGFR2 cell-based HCS assay to confirm the primary hits. The hit to lead program will be guided by using both physiologically relevant biochemical-based and cell-based assays. The combination of our novel TDA screening platform and the HCS assay technology permits us to identify unique VEGFR-2 inhibitors. Citation Format: Kelvin Lam, Edward Esposito, Deanna Navaroli, Ryan Clinton, Kerrin Beovich, Norman Garceau. Novel TDA technology and high content imaging as complementary assay platforms to identify VEGFR-2 inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4544. doi:10.1158/1538-7445.AM2013-4544

157P - Rituximab Induced Internalisation of B-Cells CD20 Receptor is Independent of the Inhibitory FC Receptor (CD32B) Intracellular Cell Signalling

ABSTRACT The anti-CD20 monoclonal antibody, rituximab, has improved treatment outcomes in B-cell malignancies. However, several B-cell lymphomas either do not respond to rituximab or develop resistance. Internalisation of rituximab may be partly responsible for this resistance. We recently showed that the level of the inhibitory Fc receptor (CD32B) at the cell surface controls the rate of rituximab internalisation. However, the precise mechanism involved has not been elucidated. Different isoforms of CD32B exist (B1 and B2), only one of which (B2) has previously been associated with an ability to internalise after engagement of immune complexes. The B1 form in contrast contains an additional intracellular region that makes it more resistant to internalisation. We therefore investigated the role of the two different CD32B isoforms (B1 and B2) and the associated intracellular tail on rituximab internalisation. CD32B1 and CD32B2 isoforms were stably transfected into CD32B-ve mouse IIA1.6 and human Ramos lymphoma cells, and flow cytometry was then used to determine the relative rates of CD32B and CD20 internalisation. Additionally, we generated mutant versions of the CD32B receptors, including those lacking the entire cytoplasmic domain to assess the importance of intracellular signalling. In contrast to expectations both B1 and B2 CD32B isoforms were downregulated upon engagement with CD32B mAb as a surrogate for immune complexes, although in agreement with earlier reports the CD32B2 isoform internalised more extensively. However, the rate of rituximab internalisation occurred equally with both isoforms and was dependent on relative expression of CD32B and the CD20:CD32B ratio at the cell surface rather than any specific activity imparted by the CD32 intracellular domain. These studies suggest that the intracellular part of CD32B is redundant for rituximab-induced CD20 internalisation and imply that internalisation is augmented by CD32B through its physical ability to bind the Fc region of rituximab at the cell surface. Disclosure All authors have declared no conflicts of interest.

Structural Determinants of Actinomyces sortase SrtC2 Required for Membrane Localization and Assembly of Type 2 Fimbriae for Interbacterial Coaggregation and Oral Biofilm Formation

As a pioneer colonizer of the oral cavity, Actinomyces oris expresses proteinaceous pili (also called fimbriae) to mediate the following two key events in biofilm formation: adherence to saliva deposits on enamel and interbacterial associations. Assembly of type 2 fimbriae that directly facilitate coaggregation with oral streptococci and Actinomyces biofilm development requires the class C sortase SrtC2. Although the general sortase-associated mechanisms have been elucidated, several structural attributes unique to the class C sortases require functional investigation. Mutational studies reported here suggest that the N-terminal transmembrane (TM) region of SrtC2, predicted to contain a signal peptide sequence, is cleaved off the mature protein and that this processing is critical for the proper integration of the enzyme at the cytoplasmic membrane, which is mediated by the extended hydrophobic C terminus containing a TM domain and a cytoplasmic tail. Deletion of this putative TM or the entire cytoplasmic domain abolished the enzyme localization and functionality. Alanine substitution of the conserved catalytic Cys-His dyad abrogated the SrtC2 enzymatic activity. In contrast, mutations designed to alter a "lid" domain that covers the catalytic pocket of a class C sortase showed no effect on enzyme activity. Finally, each of the deleterious mutations that affected SrtC2 activity or membrane localization also eliminated Actinomyces species biofilm development and bacterial coaggregation with streptococci. We conclude that the N terminus of SrtC2, which contains the signal sequence, is required for proper protein translocation and maturation, while the extended C-terminal hydrophobic region serves as a stable membrane anchor for proper enzyme functionality.

Cx50 requires an intact PDZ-binding motif and ZO-1 for the formation of functional intercellular channels

The three connexins expressed in the ocular lens each contain PDZ domain-binding motifs directing a physical association with the scaffolding protein ZO-1, but the significance of the interaction is unknown. We found that Cx50 with PDZ-binding motif mutations did not form gap junction plaques or induce cell-cell communication in HeLa cells, whereas the addition of a seven-amino acid PDZ-binding motif restored normal function to Cx50 lacking its entire C-terminal cytoplasmic domain. C-Terminal deletion had a similar although weaker effect on Cx46 but little if any effect on targeting and function of Cx43. Furthermore, small interfering RNA knockdown of ZO-1 completely inhibited the formation of gap junctions by wild-type Cx50 in HeLa cells. Thus both a PDZ-binding motif and ZO-1 are necessary for Cx50 intercellular channel formation in HeLa cells. Knock-in mice expressing Cx50 with a PDZ-binding motif mutation phenocopied Cx50 knockouts. Furthermore, differentiating lens fibers in the knock-in displayed extensive intracellular Cx50, whereas plaques in mature fibers contained only Cx46. Thus normal Cx50 function in vivo also requires an intact PDZ domain-binding motif. This is the first demonstration of a connexin-specific requirement for a connexin-interacting protein in gap junction assembly.

Endocardial cell epithelial-mesenchymal transformation requires Type III TGFβ receptor interaction with GIPC

An early event in heart valve formation is the epithelial-mesenchymal transformation (EMT) of a subpopulation of endothelial cells in specific regions of the heart tube, the endocardial cushions. The Type III TGFβ receptor (TGFβR3) is required for TGFβ2- or BMP-2-stimulated EMT in atrioventricular endocardial cushion (AVC) explants in vitro but the mediators downstream of TGFβR3 are not well described. Using AVC and ventricular explants as an in vitro assay, we found an absolute requirement for specific TGFβR3 cytoplasmic residues, GAIP-interacting protein, C terminus (GIPC), and specific Activin Receptor-Like Kinases (ALK)s for TGFβR3-mediated EMT when stimulated by TGFβ2 or BMP-2. The introduction of TGFβR3 into nontransforming ventricular endocardial cells, followed by the addition of either TGFβ2 or BMP-2, results in EMT. TGFβR3 lacking the entire cytoplasmic domain, or only the 3C-terminal amino acids that are required to bind GIPC, fails to support EMT in response to TGFβ2 or BMP-2. Overexpression of GIPC in AVC endocardial cells enhanced EMT while siRNA-mediated silencing of GIPC in ventricular cells overexpressing TGFβR3 significantly inhibited EMT. Targeting of specific ALKs by siRNA revealed that TGFβR3-mediated EMT requires ALK2 and ALK3, in addition to ALK5, but not ALK4 or ALK6. Taken together, these data identify GIPC, ALK2, ALK3, and ALK5 as signaling components required for TGFβR3-mediated endothelial cell EMT.

Targeting of mouse guanylate cyclase 1 ( Gucy2e) to Xenopus laevis rod outer segments

Photoreceptor guanylate cyclase (GC1) is a transmembrane protein and responsible for synthesis of cGMP, the secondary messenger of phototransduction. It consists of an extracellular domain, a single transmembrane domain, and an intracellular domain. It is unknown how GC1 targets to the outer segments where it resides. To identify a putative GC1 targeting signal, we generated a series of peripheral membrane and transmembrane constructs encoding extracellular and intracellular mouse GC1 fragments fused to EGFP. The constructs were expressed in Xenopus laevis rod photoreceptors under the control of the rhodopsin promoter. We examined the localization of GFP-GC1 fusion proteins containing the complete GC1 sequence, or partial GC1 sequences, which were membrane-associated via either the GC1 transmembrane domain or the rhodopsin C-terminal palmitoyl chains. Full-length GFP-GC1 targeted to the rod outer segment disk rims. As a group, fusion proteins containing the entire cytoplasmic domain of GC1 targeted to the OS, whereas other fusion proteins containing portions of the cytoplasmic or the extracellular domains did not. We conclude that GC1 likely has no single linear peptide-based OS targeting signal. Our results suggest targeting is due to either multiple weak signals in the cytoplasmic domain of GC1, or co-transport to the OS with an accessory protein.