Structure of the insulin receptor substrate IRS-1 defines a unique signal transduction protein.

Since the discovery of insulin nearly 70 years ago, there has been no problem more fundamental to diabetes research than understanding how insulin works at the cellular level. Insulin binds to the alpha subunit of the insulin receptor which activates the tyrosine kinase in the beta subunit, but the molecular events linking the receptor kinase to insulin-sensitive enzymes and transport processes are unknown. Our discovery that insulin stimulates tyrosine phosphorylation of a protein of relative molecular mass between 165,000 and 185,000, collectively called pp185, showed that the insulin receptor kinase has specific cellular substrates. The pp185 is a minor cytoplasmic phosphoprotein found in most cells and tissues; its phosphorylation is decreased in cells expressing mutant receptors defective in signalling. We have now cloned IRS-1, which encodes a component of the pp185 band. IRS-1 contains over ten potential tyrosine phosphorylation sites, six of which are in Tyr-Met-X-Met motifs. During insulin stimulation, the IRS-1 protein undergoes tyrosine phosphorylation and binds phosphatidylinositol 3-kinase, suggesting that IRS-1 acts as a multisite 'docking' protein to bind signal-transducing molecules containing Src-homology 2 and Src-homology-3 domains. Thus IRS-1 may link the insulin receptor kinase and enzymes regulating cellular growth and metabolism.     

How guanylate-binding proteins achieve assembly-stimulated processive cleavage of GTP to GMP

Interferons are immunomodulatory cytokines that mediate anti-pathogenic and anti-proliferative effects in cells. Interferon-gamma-inducible human guanylate binding protein 1 (hGBP1) belongs to the family of dynamin-related large GTP-binding proteins, which share biochemical properties not found in other families of GTP-binding proteins such as nucleotide-dependent oligomerization and fast cooperative GTPase activity. hGBP1 has an additional property by which it hydrolyses GTP to GMP in two consecutive cleavage reactions. Here we show that the isolated amino-terminal G domain of hGBP1 retains the main enzymatic properties of the full-length protein and can cleave GDP directly. Crystal structures of the N-terminal G domain trapped at successive steps along the reaction pathway and biochemical data reveal the molecular basis for nucleotide-dependent homodimerization and cleavage of GTP. Similar to effector binding in other GTP-binding proteins, homodimerization is regulated by structural changes in the switch regions. Homodimerization generates a conformation in which an arginine finger and a serine are oriented for efficient catalysis. Positioning of the substrate for the second hydrolysis step is achieved by a change in nucleotide conformation at the ribose that keeps the guanine base interactions intact and positions the beta-phosphates in the gamma-phosphate-binding site.     

GTP-binding proteins couple cardiac muscarinic receptors to a K channel

Binding of acetylcholine (ACh) to cardiac muscarinic ACh receptors (mAChR) activates a potassium channel that slows pacemaker activity. Although the time course of this activation suggests a multi-step process with intrinsic delays of 30-100 ms, no second-messenger system has been demonstrated to link the mAChR to the channel. Changes in cyclic nucleotide levels (cyclic AMP and cyclic GMP) do not affect this K channel or its response to muscarinic agonists. Indeed, electrophysiological experiments argue against the involvement of any second messenger that diffuses through the cytoplasm. We report here that coupling of the mAChR in embryonic chick atrial cells to this inward rectifying K channel requires intracellular GTP. Furthermore, pretreatment of cells with IAP (islet-activating protein from the bacterium Bordetella pertussis) eliminates the ACh-induced inward rectification. As IAP specifically ADP-ribosylates two GTP-binding proteins, Ni and No, that can interact with mAChRs, we conclude that a guanyl nucleotide-binding protein couples ACh binding to channel activation. This represents the first demonstration that a GTP-binding protein can regulate the function of an ionic channel without acting through cyclic nucleotide second messengers.     

一类图的几乎唯一泛圈性

设G是阶为n的简单Hamilton图,若存在m（3m〈n）使对每个l∈{3,4,…,n}-{m},G恰有一个长为l的圈且不含长为m的圈,则称G是几乎唯一泛圈图．用Гk^（3）表示具有n＋k条边且满足一定条件的简单外可平面的日图的集合,讨论了Гk^（3）中图的几乎唯一泛圈性．     

Isolation ofosRACD gene encoding a small GTP-binding protein from rice

Using an improved version of mRNA differential display technology, we have obtained a differentially displayed fragment RDP-8. Homologous comparison indicated that the fragment RDP-8 has high homology with the gene encoding maize small GTP-binding protein. By screening cDNA library of the rice Nongken 58N pan icle using the newly obtained fragment RDP-8 as probe, we further found the full-length cDNA of osRACD gene that encodes a rice small GTP-binding protein. Asco mpared with maize RACD gene, the osRACD of rice shows remarkable homology in both nucleotide sequence and amino acid sequence, 88% and 97% respectively. Evidence from RT-PCR study indicates that osRACD gene is related to photoperiod fertility conversion of photoperiod sensitive genic male sterility (PSGMS) rice.     

GTP-binding proteins mediate transmitter inhibition of voltage-dependent calcium channels

The modulation of voltage-dependent calcium channels by hormones and neurotransmitters has important implications for the control of many Ca2+-dependent cellular functions including exocytosis and contractility. We made use of electrophysiological techniques, including whole-cell patch-clamp recordings from dorsal root ganglion (DRG) neurones, to demonstrate a role for GTP-binding proteins (G-proteins) as signal transducers in the noradrenaline- and gamma-aminobutyric acid (GABA)-induced inhibition of voltage-dependent calcium channels. This action of the transmitters was blocked by: (1) preincubation of the cells with pertussis toxin (a bacterial exotoxin catalysing ADP-ribosylation of G-proteins); or (2) intracellular administration of guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S), a non-hydrolysable analogue of GDP that competitively inhibits the binding of GTP to G-proteins. Our findings provide the first direct demonstration of the G-protein-mediated inhibition of voltage-dependent calcium channels by neurotransmitters. This mode of transmitter action may explain the ability of noradrenaline and GABA to presynaptically inhibit Ca2+-dependent neurosecretion from DRG sensory neurones.     

Homology of 54K protein of signal-recognition particle, docking protein and two E. coli proteins with putative GTP-binding domains

Most proteins exported from mammalian cells contain a signal sequence which mediates targeting to and insertion into the membrane of the endoplasmic reticulum (ER). Involved in this process are the signal-recognition particle (SRP) and docking protein (DP), the receptor for SRP in the ER membrane. SRP interacts with the signal sequence on nascent polypeptide chains and retards their further elongation, which resumes only after interaction of the arrested ribosomal complex with the docking protein. SRP is a ribonucleoprotein particle comprising a 7S RNA and six polypeptides with relative molecular masses (Mr) of 9,000 (9K) 14K, 19K, 54K, 68K and 72K (ref. 1). The 9K and 14K proteins are essential for elongation arrest and the 68K-72K heterodimer is required for docking to the ER membrane. The 54K protein binds to the signal sequence when it emerges from the ribosome. Docking protein consists of two polypeptides, a 72K alpha-subunit (DP alpha) and a 30K beta-subunit (DP beta). No components structurally homologous to SRP and docking protein have yet been found in yeast or Escherichia coli. To understand the molecular nature of the interaction between the signal sequence and its receptor(s) we have characterized a complementary DNA coding for the 54K protein of SRP. Significant sequence homology was found to part of DP alpha and two E. coli proteins of unknown function. The homologous region includes a putative GTP-binding domain.     

Escherichia coli cell-division gene ftsZ encodes a novel GTP-binding protein.

Escherichia coli divides by forming a septum across the middle of the cell. The biochemical mechanism underlying this process is unknown. Genetic evidence suggests that of all the fts (filamentation temperature sensitive) genes involved in E. coli cell division, ftsZ plays a central role at the earliest known step of septation. Here we show that FtsZ protein binds GTP in vitro using unusual sequence elements. In contrast, such binding to the product of the conditional-lethal ftsZ84 allele is impaired. Purified FtsZ displays a Mg(2+)-dependent GTPase activity which is markedly reduced in the FtsZ84 protein. FtsZ copurifies with near stoichiometric amounts of noncovalently-bound GDP, implying the presence of a GTPase cycle in vivo, similar to that known for signal-transducing GTP-binding proteins. We also show that a small fraction of FtsZ exists as a distinct membrane-associated species that binds GTP. The membrane association of FtsZ and the known ability of GTPases to act as molecular switches implicate FtsZ in a GTP-activated signal transduction pathway that may regulate the start of septation in E. coli.     

Insulin gene enhancer binding protein Isl-1 is a member of a novel class of proteins containing both a homeo- and a Cys-His domain.

The activity of the rat insulin I gene enhancer is mainly dependent on two cis-acting protein-binding domains. Here we report the isolation of a complementary DNA encoding a protein, Isl-1, that binds to one of these domains. Isl-1 contains a homeodomain with greatest similarity to those of the Caenorhabditis elegans proteins encoded by mec-3 and lin-11. In addition, Isl-1, like the lin-11 and mec-3 gene products, contains a novel Cys-His domain which is reminiscent of known metal-binding regions. Together these proteins define a novel class of proteins containing both a homeo- and a Cys His-domain. Isl-1 is preferentially expressed in cells of pancreatic endocrine origin. If the structural homologies between Isl-1 and the C. elegans gene products reflect functional similarities, a role for Isl-1 in the development of pancreatic endocrine cells could be envisaged.     

Activation of the NADPH oxidase involves the small GTP-binding protein p21rac1

Professional phagocytes, such as neutrophils and monocytes, have an NADPH oxidase that generates superoxide and other reduced oxygen species important in killing microorganisms. Several components of the oxidase complex have been identified as targets of genetic defects causing chronic granulomatous disease. The complex consists of an electron transport chain that has as its substrate cytosolic NADPH and which discharges superoxide into the cavity of the intracellular phagocytic vacuole. The only electron transport component identified so far is a low-potential cytochrome b, apparently the only membrane component required. At least three cytosolic factors are also necessary, two of which, p67phOx and p47phOx, have been identified by their absence in patients with chronic granulomatous disease. A third component, sigma 1, is required for stimulation of oxidase activity in a cell-free system. The active components of purified sigma 1 are two proteins that associate as heterodimers, and here we report that these are the small GTP-binding protein p21rac1 and the GDP-dissociation inhibitor rhoGDI.     

Farnesylated gamma-subunit of photoreceptor G protein indispensable for GTP-binding

Transducin, composed of subunits T alpha, T beta and T gamma, is a member of a heterotrimeric G-protein family, and transduces the light signal in visual cells. We have recently found that bovine T beta gamma can be separated into two components. T beta gamma-1 and T beta gamma-2, each of which has its own gamma-subunit, T gamma-1 and T gamma-2, respectively. T beta gamma-2 enhances the binding of GTP to T alpha in the presence of metarhodopsin II by about 30-fold compared with T beta gamma-1. Here we show that a farnesyl moiety is attached to a sulphur atom of the C-terminal cysteine of T gamma-2 (active form), a part of which is additionally methyl-esterified at the alpha-carboxyl group. In T gamma-1 (inactive form), however, such modifications are missing. Thus, the farnesyl moiety attached to the gamma-subunit is indispensable for the GTP-binding activity of transducin. This suggests that a similar modification may occur in the gamma-subunits of other heterotrimeric G proteins involved in biological signal transduction processes.     

Insulin-regulatable tissues express a unique insulin-sensitive glucose transport protein

At least three different glucose transport systems exist in mammalian cells. These are: (1) the constitutively active, facilitative carrier characteristic of human erythrocytes, Hep G2 (ref. 2) cells and rat brain; (2) the Na-dependent active transporter of kidney and small intestine; and (3) the facilitative carrier of rat liver (B. Thorens and H. F. Lodish, personal communication). A fourth possible glucose transport system is the insulin-dependent carrier that may be specific to muscle and adipose tissue. This transporter resides primarily in an intracellular compartment in resting cells from where it translocates to the cell surface upon cellular insulin exposure. This raises the question of whether hormonal regulation of glucose transport is conferred by virtue of a tissue-specific signalling mechanism or a tissue-specific glucose transporter. Here we present data supporting the latter concept based upon a monoclonal antibody against the fat cell glucose transporter that identifies a unique, insulin-regulatable glucose transport protein in muscle and adipose tissue.     

Molecular cloning of the microtubule-associated mechanochemical enzyme dynamin reveals homology with a new family of GTP-binding proteins

A complementary DNA encoding the D100 polypeptide of rat brain dynamin--a force-producing, microtubule-activated nucleotide triphosphatase--has been cloned and sequenced. The predicted amino acid sequence includes a guanine nucleotide-binding domain that is homologous with those of a family of antiviral factors, inducible by interferon and known as Mx proteins, and with the product of the essential yeast vacuolar protein sorting gene VPS1. These relationships imply the existence of a new family of GTPases with physiological roles that may include microtubule-based motility and protein sorting.     

Structure of the human class I histocompatibility antigen, HLA-A2

The class I histocompatibility antigen from human cell membranes has two structural motifs: the membrane-proximal end of the glycoprotein contains two domains with immunoglobulin-folds that are paired in a novel manner, and the region distal from the membrane is a platform of eight antiparallel beta-strands topped by alpha-helices. A large groove between the alpha-helices provides a binding site for processed foreign antigens. An unknown 'antigen' is found in this site in crystals of purified HLA-A2.     

Unexpected expression of a unique mixed-isotype class II MHC molecule by transfected L-cells

Class II (Ia) major histocompatibility complex (MHC) molecules are heterodimeric integral membrane proteins composed of non-covalently linked alpha and beta glycoprotein chains. Studies of both normal cells and L-cell transfectants have shown that neither alpha- nor beta-chains are found on the cell surface alone, and that alpha beta dimers are required for membrane expression. In both mouse and man, several distinct non-allelic alpha and beta genes exist. Analysis of Ia molecules by immunoprecipitation and two-dimensional gel electrophoresis has demonstrated apparently selective association of particular pairs of the various alpha- and beta-chains to form the expressed class II isotypes I-A and I-E (mouse) or DQ, DP and DR (human). Because the various alpha- or beta-chains encoded by distinct loci exist in many allelic forms within a species, such specific pairing suggests a special role for isotypically conserved regions of each chain in the association process. In attempting to localize such putative assembly-controlling regions using the technique of DNA-mediated gene transfer, various combinations of murine alpha and beta genes were introduced into L-cells. Here we report the unexpected observation, following transfection, of mixed-isotype (Ad beta Ea/k alpha) molecules on the L-cell membrane and document that the formation of this pair is strongly influenced by allelic polymorphism of the A beta chain.     

HIV F/3' orf encodes a phosphorylated GTP-binding protein resembling an oncogene product

Apart from the retroviral gag, pol and env the HIV genome contains the F (3' orf) gene which encodes a polypeptide of 206 amino acids which is myristylated at the N-terminal and whose function is unknown. We have expressed the F gene in Escherichia coli and from a recombinant vaccinia virus, VVTGfHIV. The F-protein produced in VVTGfHIV-infected mammalian cells is myristilated, and is phosphorylated by protein kinase C at a residue close to the N-terminus like pp60-src (ref. 5). Purified bacterial F-protein also shows the GTPase, autophosphorylation and GTP-binding activities reported for the ras gene product. Furthermore, we show that expression of F in a CD4+ cell line down-regulates the CD4(T4) antigen. These results suggest that F is important in the pathophysiology of AIDS (acquired immune deficiency syndrome).     

一类复Gross-Pitaevskii方程的H2 解的唯一存在性

利用Galerkin方法和Leray-Schauder不动点定理,一类复Gross-Pitaevskii方程的初边值问题的H2解的唯一存在性得到了证明.     

一类单滤过李代数的结构

研究单的滤过李代数,使其相联阶化李代数同构于李代数Gn,得到这样的滤过李代数也同构于Gn。