Cytoplasmic Domain Interactions Research Articles

14-3-3zeta Regulates Transmembrane Receptor Signaling by Binding TRAF Proteins

Abstract Adaptor proteins are critical for signal transduction events involving cytokine signaling, host-response, and autoimmune diseases. Our laboratory is interested in understanding the immune regulatory role of 14-3-3zeta (z), an adaptor protein, in signal transductions and diseases. We examined the role of 14-3-3z in interleukin-17 (IL-17A) signaling and autoimmune inflammatory arthritis using genetically modified cells and rats. The IL-17A signal transduction requires IL-17 receptor cytoplasmic domain interactions with Act1 and several TRAF proteins. Our results show that 14-3-3z interacts with TRAF6, TRAF2, and TRAF5 upon IL-17A stimulation which has functional consequences. The interaction of 14-3-3z and TRAF protein was found to be specific, as mutation of the interacting residues resulted in the loss of binding with functional impacts. Further interaction studies show a higher affinity of 14-3-3z for TRAF6, which has two putative 14-3-3 binding sites. To examine the generality of 14-3-3z-TRAF interactions and their role in another transmembrane receptor signaling, we examined RANKL signal transduction. Similar to IL-17A, 14-3-3z-TRAF interaction was increased upon RANKL treatment and had functional consequences. While there are common domains and significant sequence conservation between the TRAF proteins, a specificity of interactions exists with 14-3-3z. Our results show that the 14-3-3z-TRAF axis is a novel regulatory mechanism underneath several transmembrane receptor-mediated signaling. Current studies target understanding the molecular basis of interactions between 14-3-3z and specific TRAF proteins.

Identification of Inhibitors of Integrin Cytoplasmic Domain Interactions With Syk.

Leukocyte inflammatory responses require integrin cell-adhesion molecule signaling through spleen tyrosine kinase (Syk), a non-receptor kinase that binds directly to integrin β-chain cytoplasmic domains. Here, we developed a high-throughput screen to identify small molecule inhibitors of the Syk-integrin cytoplasmic domain interactions. Screening small molecule compound libraries identified the β-lactam antibiotics cefsulodin and ceftazidime, which inhibited integrin β-subunit cytoplasmic domain binding to the tandem SH2 domains of Syk (IC50 range, 1.02–4.9 µM). Modeling suggested antagonist binding to Syk outside the pITAM binding site. Ceftazidime inhibited integrin signaling via Syk, including inhibition of adhesion-dependent upregulation of interleukin-1β and monocyte chemoattractant protein-1, but did not inhibit ITAM-dependent phosphorylation of Syk mediated by FcγRI signaling. Our results demonstrate a novel means to target Syk independent of its kinase and pITAM binding sites such that integrin signaling via this kinase is abrogated but ITAM-dependent signaling remains intact. As integrin signaling through Syk is essential for leukocyte activation, this may represent a novel approach to target inflammation.

Impaired cytoplasmic domain interactions cause co-assembly defect and loss of function in the p.Glu293Lys KNCJ2 variant isolated from an Andersen-Tawil syndrome patient.

Subunit interactions at the cytoplasmic domain interface (CD-I) have recently been shown to control gating in inward rectifier potassium channels. Here we report the novel KCNJ2 variant p.Glu293Lys that has been found in a patient with Andersen-Tawil syndrome type 1 (ATS1), causing amino acid substitution at the CD-I of the inward rectifier potassium channel subunit Kir2.1. Neither has the role of Glu293 in gating control been investigated nor has a pathogenic variant been described at this position. This study aimed to assess the involvement of Glu293 in CD-I subunit interactions and to establish the pathogenic role of the p.Glu293Lys variant in ATS1. The p.Glu293Lys variant produced no current in homomeric form and showed dominant-negative effect over wild-type (WT) subunits. Immunocytochemical labelling showed the p.Glu293Lys subunits to distribute in the subsarcolemmal space. Salt bridge prediction indicated the presence of an intersubunit salt bridge network at the CD-I of Kir2.1, with the involvement of Glu293. Subunit interactions were studied by the NanoLuc® Binary Technology (NanoBiT) split reporter assay. Reporter constructs carrying NanoBiT tags on the intracellular termini produced no bioluminescent signal above background with the p.Glu293Lys variant in homomeric configuration and significantly reduced signals in cells co-expressing WT and p.Glu293Lys subunits simultaneously. Extracellularly presented reporter tags, however, generated comparable bioluminescent signals with heteromeric WT and p.Glu293Lys subunits and with homomeric WT channels. Loss of function and dominant-negative effect confirm the causative role of p.Glu293Lys in ATS1. Co-assembly of Kir2.1 subunits is impaired in homomeric channels consisting of p.Glu293Lys subunits and is partially rescued in heteromeric complexes of WT and p.Glu293Lys Kir2.1 variants. These data point to an important role of Glu293 in mediating subunit assembly, as well as in gating of Kir2.1 channels.

Substrate Specificity of the FurE Transporter Is Determined by Cytoplasmic Terminal Domain Interactions.

FurE, a member of the Nucleobase Cation Symporter 1 transporter family in Aspergillus nidulans, is specific for allantoin, uric acid (UA), uracil, and related analogs. Herein, we show that C- or N-terminally-truncated FurE transporters (FurE-ΔC or FurE-ΔΝ) present increased protein stability, but also an inability for UA transport. To better understand the role of cytoplasmic terminal regions, we characterized genetic suppressors that restore FurE-ΔC-mediated UA transport. Suppressors map in the periphery of the substrate-binding site [Thr133 in transmembrane segment (TMS)3 and Val343 in TMS8], an outward-facing gate (Ser296 in TMS7, Ile371 in TMS9, and Tyr392 and Leu394 in TMS10), or in flexible loops (Asp26 in LN, Gly222 in L5, and Asn308 in L7). Selected suppressors were also shown to restore the wild-type specificity of FurE-ΔΝ, suggesting that both C- and/or N-terminal domains are involved in intramolecular dynamics critical for substrate selection. A direct, substrate-sensitive interaction of C- and/or N-terminal domains was supported by bimolecular fluorescence complementation assays. To our knowledge, this is the first case where not only the function, but also the specificity, of a eukaryotic transporter is regulated by its terminal cytoplasmic regions.

TβRIII independently binds type I and type II TGF-β receptors to inhibit TGF-β signaling.

Transforming growth factor-β (TGF-β) receptor oligomerization has important roles in signaling. Complex formation among type I and type II (TβRI and TβRII) TGF-β receptors is well characterized and is essential for signal transduction. However, studies on their interactions with the type III TGF-β coreceptor (TβRIII) in live cells and their effects on TGF-β signaling are lacking. Here we investigated the homomeric and heteromeric interactions of TβRIII with TβRI and TβRII in live cells by combining IgG-mediated patching/immobilization of a given TGF-β receptor with fluorescence recovery after photobleaching studies on the lateral diffusion of a coexpressed receptor. Our studies demonstrate that TβRIII homo-oligomerization is indirect and depends on its cytoplasmic domain interactions with scaffold proteins (mainly GIPC). We show that TβRII and TβRI bind independently to TβRIII, whereas TβRIII augments TβRI/TβRII association, suggesting that TβRI and TβRII bind to TβRIII simultaneously but not as a complex. TβRIII expression inhibited TGF-β-mediated Smad2/3 signaling in MDA-MB-231 cell lines, an effect that depended on the TβRIII cytoplasmic domain and did not require TβRIII ectodomain shedding. We propose that independent binding of TβRI and TβRII to TβRIII competes with TβRI/TβRII signaling complex formation, thus inhibiting TGF-β-mediated Smad signaling.

Crystals of Na+/K+-ATPase with bound cisplatin

Cisplatin is the most widely used chemotherapeutics for cancer treatment, however, its administration is connected to inevitable adverse effects. Previous studies suggested that cisplatin is able to inhibit Na+/K+-ATPase (NKA), the enzyme responsible for maintaining electrochemical potential and sodium gradient across the plasma membrane. Here we report a crystallographic analysis of cisplatin bound to NKA in the ouabain bound E2P form. Despite a moderate resolution (7.4Å and 7.9Å), the anomalous scattering from platinum and a model representation from a recently published structure enabled localization of seven cisplatin binding sites by anomalous difference Fourier maps. Comparison with NKA structures in the E1P conformation suggested two possible inhibitory mechanisms for cisplatin. Binding to Met151 can block the N-terminal pathway for transported cations, while binding to Met171 can hinder the interaction of cytoplasmic domains during the catalytic cycle.

Functional Antagonism of Rhesus Macaque and Chimpanzee BST-2 by HIV-1 Vpu Is Mediated by Cytoplasmic Domain Interactions

Human immunodeficiency virus type 1 (HIV-1) Vpu enhances the release of viral particles from infected cells by interfering with the function of BST-2/tetherin, a cellular protein inhibiting virus release. The Vpu protein encoded by NL4-3, a widely used HIV-1 laboratory strain, antagonizes human BST-2 but not monkey or murine BST-2, leading to the conclusion that BST-2 antagonism by Vpu is species specific. In contrast, we recently identified several primary Vpu isolates, such as Vpu of HIV-1DH12, capable of antagonizing both human and rhesus BST-2. Here we report that while Vpu interacts with human BST-2 primarily through their respective transmembrane domains, antagonism of rhesus BST-2 by Vpu involved an interaction of their cytoplasmic domains. Importantly, a Vpu mutant carrying two mutations in its transmembrane domain (A14L and W22A), rendering it incompetent for interaction with human BST-2, was able to interact with human BST-2 carrying the rhesus BST-2 cytoplasmic domain and partially neutralized the ability of this BST-2 variant to inhibit viral release. Bimolecular fluorescence complementation analysis to detect Vpu-BST-2 interactions suggested that the physical interaction of Vpu with rhesus or chimpanzee BST-2 involves a 5-residue motif in the cytoplasmic domain of BST-2 previously identified as important for the antagonism of monkey and great ape BST-2 by simian immunodeficiency virus (SIV) Nef. Thus, our study identifies a novel mechanism of antagonism of monkey and great ape BST-2 by Vpu that targets the same motif in BST-2 used by SIV Nef and might explain the expanded host range observed for Vpu isolates in our previous study.

The Hyaluronan Receptor for Endocytosis (HARE) Activates NF-κB-mediated Gene Expression in Response to 40–400-kDa, but Not Smaller or Larger, Hyaluronans

The hyaluronan (HA) receptor for endocytosis (HARE; Stabilin-2) binds and clears 14 different ligands, including HA and heparin, via clathrin-mediated endocytosis. HA binding to HARE stimulates ERK1/2 activation (Kyosseva, S. V., Harris, E. N., and Weigel, P. H. (2008) J. Biol. Chem. 283, 15047-15055). To assess a possible HA size dependence for signaling, we tested purified HA fractions of different weight-average molar mass and with narrow size distributions and Select-HA(TM) for stimulation of HARE-mediated gene expression using an NF-κB promoter-driven luciferase reporter system. Human HARE-mediated gene expression was stimulated in a dose-dependent manner with small HA (sHA) >40 kDa and intermediate HA (iHA) <400 kDa. The hyperbolic dose response saturated at 20-50 nM with an apparent K(m) ~10 nM, identical to the Kd for HA-HARE binding. Activation was not detected with oligomeric HA (oHA), sHA <40 kDa, iHA >400 kDa, or large HA (lHA). Similar responses occurred with rat HARE. Activation by sHA-iHA was blocked by excess nonsignaling sHA, iHA, or lHA, deletion of the HA-binding LINK domain, or HA-blocking antibody. Endogenous NF-κB activation also occurred in the absence of luciferase plasmids, as assessed by degradation of IκB-α. ERK1/2 activation was also HA size-dependent. The results show that HA-HARE interactions stimulate NF-κB-activated gene expression and that HARE senses a narrow size range of HA degradation products. We propose a model in which optimal length HA binds multiple HARE proteins to allow cytoplasmic domain interactions that stimulate intracellular signaling. This HARE signaling system during continuous HA clearance could monitor the homeostasis of tissue biomatrix turnover throughout the body.

Hereditary red cell membrane defects: diagnostic and clinical aspects.

The plasma membrane of the erythrocyte accounts for all of this cell’s antigenic, transport, and mechanical characteristics, particularly its ability to undergo large passive deformations during repeated passage through the narrow capillaries of the microvasculature, throughout its 120-day life span. The determinant of normal membrane cohesion is the system of “vertical” linkages between the phospholipid bilayer and membrane skeleton, formed by the interactions of the cytoplasmic domains of various membrane proteins with the spectrin-based skeletal network. Band 3 and Rh-associated glycoprotein (RhAG) provide such links by interacting with ankyrin, which in turn binds to β-spectrin. Protein 4.2 binds to both band 3 and ankyrin and can regulate the avidity of the interaction between band 3 and ankyrin. Glycophorin C, band 3, XK, Rh, and Duffy all bind to protein 4.1R, the third member of the ternary junctional complex with β-spectrin and actin1–2. Red cell membrane disorders are inherited diseases due to mutations in various membrane or skeletal proteins, resulting in decreased red cell deformability, reduced life span and premature removal of the erythrocytes from the circulation. The red cell membrane disorders include hereditary spherocytosis, hereditary elliptocytosis, hereditary ovalocytosis and hereditary stomatocytosis.

Partial unfolding and oligomerization of stromal interaction molecules as an initiation mechanism of store operated calcium entryThis paper is one of a selection of papers published in this special issue entitled “Canadian Society of Biochemistry, Molecular &amp; Cellular Biology 52nd Annual Meeting — Protein Folding: Principles and Diseases” and has undergone the Journal's usual peer review process.

Spatiotemporally discrete cytoplasmic Ca2+ fluctuations are fundamental eukaryotic signals in myriad physiological and pathophysiological functions. Store-operated Ca2+ entry is the process whereby a decrease in endoplasmic reticulum (ER) luminal Ca2+ levels activates Ca2+ release activated calcium (CRAC) channels on the plasma membrane (PM), providing a sustained Ca2+ elevation to the cytoplasm and ultimately replenishing the ER lumen Ca2+ supply. Stromal interaction molecules (STIMs) are the Ca2+ sensors of the ER lumen, which macromolecularly couple depleted ER Ca2+ to the assembly and opening of PM CRAC channels. The considerable stability difference caused by Ca2+ loading and depletion within the luminal portion of STIMs modulates intramolecular cytoplasmic domain interactions essential to the assembly of PM CRAC channels. Thus, the action of the entire complex is tightly regulated through the Ca2+ sensitivity of luminal STIM domains. Recent structural and biochemical studies suggest that partial unfolding - coupled oligomerization of STIMs is a crucial step in CRAC channel activation. Based on these and other published data, this minireview discusses what is currently known about the molecular mechanism of ER Ca2+ sensing by STIMs.

Protein-Protein and Protein-Lipid Interactions Modulate αIIbβ3 Inside-out Signaling.

Abstract 152Integrins are ubiquitously expressed α/β heterodimers that mediate cell-cell and cell-extracellular matrix interactions. The platelet integrin αIIbβ3 binds soluble fibrinogen following platelet activation, an event necessary for the formation of platelet aggregates. Integrins reside on plasma membranes in a highly regulated and dynamic equilibrium between inactive resting states and active ligand binding conformations. An essential feature of this equilibrium is the association and dissociation of integrin transmembrane (TM) and cytoplasmic domains. Thus, when integrins are inactive, the TM and cytoplasmic domains of their α and β subunits are in proximity; the domains separate when integrins assume their active conformations. Agonist-induced activation of αIIbβ3 in platelets (inside-out activation) requires binding of the FERM domain of the cytoskeletal protein talin to highly conserved membrane proximal and distal sequences in the β3 subunit cytoplasmic domain. FERM domain binding to the β3 cytoplasmic domain likely disrupts the αIIbβ3 TM domain-cytoplasmic domain heterodimer. As the β3 cytoplasmic domain exists in close apposition to the inner leaflet of the plasma membrane and the talin FERM domain is known to interact with the negatively-charged phospholipids that are common in eukaryotic membranes, we have investigated the effects of the membrane environment on β3 and talin structure and the talin-β;3 protein-protein interaction that regulates αIIbβ3 activation. A water soluble form of the β3 cytoplasmic domain was expressed, purified, and chemically conjugated to phospholipid bilayers, mimicking the native environment of the αIIb and β3 cytoplasmic domains as they exit the membrane. Using circular dichroism (CD) spectroscopy, the β3 cytoplasmic domain was found to have a significant increase in secondary structure and overall helicity when attached to the bilayer. Similarly, using hydrogen-deuterium exchange (HDX) mass spectrometry, we found that there is an overall decrease in amide backbone flexibility, particularly in the membrane proximal talin-interacting region. We also found significant changes in talin secondary structure in detergents by CD and by limited proteolysis mapping, supporting dramatic conformational changes in talin upon membrane binding. Using isothermal titration calorimetry (ITC) and integrin-conjugated lipid bilayers, we investigated the effects of lipid composition on talin-bilayer and talin-β3 cytoplasmic domain interactions. We found that the talin FERM domain likely has two binding sites for the membrane-conjugated β3 cytoplasmic domain, one in the canonical F3 subdomain and a second that depends on the F2 subdomain. Moreover, the talin head domain does not bind to β3 on neutral bilayers and requires a significant excess of phosphatidylserine (PS) relative to β3 to allow saturation. Interestingly phosphatidylinositol (4, 5)-bisphosphate containing bilayers have significantly higher affinity and mitigate the need for excess PS. Our results point toward a model in which β3 cytoplasmic domain structure is dependent on the lipid environment and its interaction with talin. Thus, αIIbβ3 activation is significantly regulated by lipid which may play a role in constraining αIIbβ3 activation by talin in the crowded platelet intracellular environment. Disclosures:No relevant conflicts of interest to declare.

2P-024 SH3ドメイン欠損変異体を用いたGrb2及びGadsとCD28細胞質内ドメインとの分子間相互作用(蛋白質-構造機能相関,第47回日本生物物理学会年会)

2P-024 SH3ドメイン欠損変異体を用いたGrb2及びGadsとCD28細胞質内ドメインとの分子間相互作用(蛋白質-構造機能相関,第47回日本生物物理学会年会)

Proteomics of Bovine Myelin Sheath: Characterization of a Truncated Form of P0 by MALDI-TOF/TOF Mass Spectrometry

The glycoprotein P0, the major structural protein of the peripheral nerve myelin, plays a critical role in holding myelin lamellae together via interaction of both extracellular and cytoplasmic domains. Mutations in the human P0 gene give rise to severe and progressive forms of dominantly inherited peripheral neuropathies like CMT1B. Here we report on the characterization of a bovine P0-derived protein of nearly 26 kD that corresponds to the P0 protein truncated in its cytoplasmic domain. Matrix assisted laser desorption ionization (MALDI)-time-of-flight/time-of-flight (TOF/TOF) mass spectrometry (MS) analysis on its tryptic digest has provided a peptide mapping, the main difference of which from the normal P0 analog was represented by the absence of the cluster of peaks at m/z 1513.7501, 1530.7701, and 1546.7651. The latter corresponds to the P0 fragment QTPVLYAMLDHSR and to its pyroglutamic and methionine-oxidized derivatives. The species at 1530.7701 covering the sequence 186-198 of P0 is not an artifact and might have a functional role in the myelin architecture.

Adaptor heat shock protein complex formation regulates trafficking of the asialoglycoprotein receptor

In the asialoglycoprotein receptor (ASGPR) endocytic pathway, internalized receptors pass through early, recycling, and sorting endosomal compartments before returning to the cell surface. Sorting motifs in the cytoplasmic domain (CD) and protein interactions with these sequences presumably direct receptor trafficking. Previous studies have shown that association of a potential sorting heat shock protein (HSP) heterocomplex with the ASGPR-CD was regulated by casein kinase 2 (CK2)-mediated phosphorylation. Mass spectrometry and immunoblot analyses identified five of these ASGPR-CD-associated proteins as the molecular chaperones glycoprotein 96, HSP70, HSP90, cyclophilin A, and FK 506 binding protein. The present study was undertaken to determine whether any of the adaptor protein complexes (AP1, AP2, or AP3) were selectivity associated with the ASGPR-CD. In conjunction with molecular chaperones, AP2 and AP1 were recovered from a CK2 phosphorylated agarose-GSH-GST-ASGPR-CD matrix. Binding of AP3 was independent of the phosphorylation status of the CD matrix. Inhibition of CK2-mediated phosphorylation with tetrabromobenzotriazole prevented AP recovery within an immunoadsorbed ASGPR complex. Rapamycin, which dissociates the HSP heterocomplex from ASGPR-CD, thereby altering receptor trafficking also, inhibited AP association. Similar results were obtained with an inhibitor of HSP90 heterocomplex formation, geldanmycin. The data presented provide evidence that recruitment of AP1 and AP2, which is necessary for appropriate receptor trafficking, is mediated by the interaction of AP with the ASGPR-CD-bound HSP complex.

Integrin β cytoplasmic domain interactions with phosphotyrosine-binding domains: A structural prototype for diversity in integrin signaling

The cytoplasmic domains (tails) of heterodimeric integrin adhesion receptors mediate integrins' biological functions by binding to cytoplasmic proteins. Most integrin beta tails contain one or two NPXYF motifs that can form beta turns. These motifs are part of a canonical recognition sequence for phosphotyrosine-binding (PTB) domains, protein modules that are present in a wide variety of signaling and cytoskeletal proteins. Indeed, talin and ICAP1-alpha bind to integrin beta tails by means of a PTB domain-NPXY ligand interaction. To assess the generality of this interaction we examined the binding of a series of recombinant PTB domains to a panel of short integrin beta tails. In addition to the known integrin-binding proteins, we found that Numb (a negative regulator of Notch signaling) and Dok-1 (a signaling adaptor involved in cell migration) and their isolated PTB domain bound to integrin tails. Furthermore, Dok-1 physically associated with integrin alpha IIb beta 3. Mutations of the integrin beta tails confirmed that these interactions are canonical PTB domain-ligand interactions. First, the interactions were blocked by mutation of an NPXY motif in the integrin tail. Second, integrin class-specific interactions were observed with the PTB domains of Dab, EPS8, and tensin. We used this specificity, and a molecular model of an integrin beta tail-PTB domain interaction to predict critical interacting residues. The importance of these residues was confirmed by generation of gain- and loss-of-function mutations in beta 7 and beta 3 tails. These data establish that short integrin beta tails interact with a large number of PTB domain-containing proteins through a structurally conserved mechanism.

Structure and mechanism of Na,K-ATPase: functional sites and their interactions.

The cell membrane Na,K-ATPase is a member of the P-type family of active cation transport proteins. Recently the molecular structure of the related sarcoplasmic reticulum Ca-ATPase in an E1 conformation has been determined at 2.6 A resolution. Furthermore, theoretical models of the Ca-ATPase in E2 conformations are available. As a result of these developments, these structural data have allowed construction of homology models that address the central questions of mechanism of active cation transport by all P-type cation pumps. This review relates recent evidence on functional sites of Na,K-ATPase for the substrate (ATP), the essential cofactor (Mg(2+) ions), and the transported cations (Na(+) and K(+)) to the molecular structure. The essential elements of the Ca-ATPase structure, including 10 transmembrane helices and well-defined N, P, and A cytoplasmic domains, are common to all PII-type pumps such as Na,K-ATPase and H,K-ATPases. However, for Na,K-ATPase and H,K-ATPase, which consist of both alpha- and beta-subunits, there may be some detailed differences in regions of subunit interactions. Mutagenesis, proteolytic cleavage, and transition metal-catalyzed oxidative cleavages are providing much evidence about residues involved in binding of Na(+), K(+), ATP, and Mg(2+) ions and changes accompanying E1-E2 or E1-P-E2-P conformational transitions. We discuss this evidence in relation to N, P, and A cytoplasmic domain interactions, and long-range interactions between the active site and the Na(+) and K(+) sites in the transmembrane segments, for the different steps of the catalytic cycle.

The kiss of death: promises and failures of death receptors and ligands in cancer therapy.

Death receptors and their ligands exert important regulatory functions in the maintenance of tissue homeostasis and the physiological regulation of programmed cell death. Currently, six different death receptors are known including tumor necrosis factor (TNF) receptor-1, CD95 (Fas/APO-1), TNF receptor-related apoptosis-mediating protein (TRAMP), TNF-related apoptosis-inducing ligand (TRAIL) receptor-1 and -2, and death receptor-6 (DR6). The signaling pathways by which these receptors induce apoptosis are similar and rely on oligomerization of the receptor by death ligand binding, recruitment of an adapter protein through homophilic interaction of cytoplasmic domains, and subsequent activation of an inducer caspase which initiates execution of the cell death programme. The ability of these receptors and their ligands to kill malignant cells was discovered early and helped to coin the term 'tumor necrosis factor' for the first identified death ligand. This review summarizes the current and rapidly expanding knowledge about the signaling pathways triggered by death receptor/ligand systems, their potency in experimental cancer therapy, and their therapeutic limitations, especially regarding their toxicity for non-malignant cells.

The Energy Transduction Mechanism of Na,K-ATPase Studied with Iron-catalyzed Oxidative Cleavage

This paper extends our recent report on specific iron-catalyzed oxidative cleavages of renal Na,K-ATPase and effects of E1 left arrow over right arrow E2 conformational transitions (Goldshleger, R. , and Karlish, S. J. D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9596-9601). The experiments indicate that only peptide bonds close to a bound Fe2+ ion are cleaved, and provide evidence on proximity of the different cleavage positions in the native enzyme. A sequence HFIH near trans-membrane segment M3 appears to be involved in Fe2+ binding. Previously we hypothesized that E2 and E1 conformations are characterized by formation or relaxation of interactions within the alpha subunit at or near highly conserved sequences, TGES in the minor cytoplasmic loop and CSDK, MVTGD, and VNDSPALKK in the major cytoplasmic loop. This concept has been tested by examining iron-catalyzed cleavage in both non-phosphorylated and phosphorylated conformations and effects of phosphate, vanadate, and ouabain. The results imply that both E1 left arrow over right arrow E2 and E1P left arrow over right arrow E2P transitions are indeed associated with formation and relaxation of interactions between cytoplasmic domains, comprising the minor loop plus N-terminal tail leading into M1 and major loop, respectively. Furthermore, it appears that either non-covalently or covalently bound phosphate bind near CSDK and MVTGD, and Mg2+ ions may bind to residues within TGES and VNDSPALKK and to bound phosphate. Thus cytoplasmic domain interactions seem to occur within or near the active site. We discuss the relationship between structural changes in the cytoplasmic domain and movements of trans-membrane segments that lead to cation transport. Presumably conformation-dependent formation and relaxation of domain interactions underlie energy transduction in all P-type pumps.

Functional Interactions of Cytoplasmic Domains of the Skeletal Muscle Ca 2+ Release Channel

The skeletal muscle Ca 2+ release channel (RYR1) is a homotetramer with subunits of 565 kD. Although the channel part of this protein is probably formed by the C-terminal one fifth of the protein, most of the regulation of channel activity is likely to arise from intermolecular and intramolecular interactions of its large cytoplasmic domain. This cytoplasmic region of the protein is thought to contain binding sites for a variety of modulators, including the t-tubule voltage sensor, and mutations in some cases of malignant hyperthermia and central core disease. Regulation of channel activity must, therefore, involve long-distance allosteric modulation arising from changes in both intersubunit and intrasubunit interactions within the cytoplasmic domain of the RYR1 tetramer. Biochemical studies are beginning to elucidate some of the sites within the cytoplasmic domains important for modulting channel activity in the membrane-spanning domain. This review summarizes these findings and presents a working model for the regulation of the channel by the interactions of its cytoplasmic domains.

A Conserved Sequence Motif in the Integrin β3 Cytoplasmic Domain Is Required for Its Specific Interaction with β3-Endonexin

Integrin signaling is mediated by interaction of integrin cytoplasmic domains with intracellular signaling molecules. Recently, we identified a novel 111-amino acid polypeptide, termed beta3-endonexin, which interacts selectively with the integrin beta3 cytoplasmic domain. In the present study we conducted a systematic mutational analysis of both the integrin beta3 cytoplasmic domain and beta3-endonexin to map sites required for interaction. The interaction of the full-length beta3 integrin subunit with beta3-endonexin in vitro required the beta3 cytoplasmic domain. In a yeast two-hybrid system, both membrane-proximal and membrane-distal residues of the beta3 cytoplasmic domain were necessary for interaction with beta3-endonexin. In particular, the membrane-distal NITY motif at beta3 756-759 was critical for the interaction. Exchange of beta3 residues 756-759 (NITY) for the corresponding residues in beta1 (NPKY) endowed the beta1 cytoplasmic domain with the ability to interact with beta3-endonexin. Conversely, exchange of the NPKY motif at beta1 772-775 for the NITY motif in beta3 abolished interaction of this chimeric cytoplasmic domain with beta3-endonexin. Because the NITY motif is present in the beta3 but not the beta1 cytoplasmic domain, these results explain the selective interaction of this cytoplasmic domain with beta3-endonexin. In addition, deletional analysis suggested that a core 91-residue sequence of beta3-endonexin is sufficient for specific binding to the beta3 cytoplasmic domain. These studies have identified a cytoplasmic domain sequence motif that specifies an integrin-specific protein-protein interaction.