Development of the signal in sensory rhodopsin and its transfer to the cognate transducer

The microbial phototaxis receptor sensory rhodopsin II (NpSRII, also named phoborhodopsin) mediates the photophobic response of the haloarchaeon Natronomonas pharaonis by modulating the swimming behaviour of the bacterium. After excitation by blue-green light NpSRII triggers, by means of a tightly bound transducer protein (NpHtrII), a signal transduction chain homologous with the two-component system of eubacterial chemotaxis. Two molecules of NpSRII and two molecules of NpHtrII form a 2:2 complex in membranes as shown by electron paramagnetic resonance and X-ray structure analysis. Here we present X-ray structures of the photocycle intermediates K and late M (M2) explaining the evolution of the signal in the receptor after retinal isomerization and the transfer of the signal to the transducer in the complex. The formation of late M has been correlated with the formation of the signalling state. The observed structural rearrangements allow us to propose the following mechanism for the light-induced activation of the signalling complex. On excitation by light, retinal isomerization leads in the K state to a rearrangement of a water cluster that partly disconnects two helices of the receptor. In the transition to late M the changes in the hydrogen bond network proceed further. Thus, in late M state an altered tertiary structure establishes the signalling state of the receptor. The transducer responds to the activation of the receptor by a clockwise rotation of about 15 degrees of helix TM2 and a displacement of this helix by 0.9 A at the cytoplasmic surface.     

Peptide chemotaxis in E. coli involves the Tap signal transducer and the dipeptide permease

Bacterial chemotaxis provides a simple model system for the more complex sensory responses of multicellular eukaryotic organisms. In Escherichia coli, methylation and demethylation of four related membrane proteins, the methyl-accepting chemotaxis proteins (or MCPs), is central to chemotactic sensing and signal transduction. Three of these proteins, Tar, Tsr and Trg, have been assigned specific roles in chemotaxis. However, the role of the fourth MCP, Tap, has remained obscure. We demonstrate here that Tap functions as a conventional signal transducer, enabling the cell to respond chemotactically to dipeptides. This provides the first evidence of specific bacterial chemotaxis towards peptides. Peptide taxis requires the function of a periplasmic component of the dipeptide permease. This protein represents the first example of a periplasmic chemoreceptor that does not have a sugar substrate.     

Four-helical-bundle structure of the cytoplasmic domain of a serine chemotaxis receptor.

The bacterial chemotaxis receptors are transmembrane receptors with a simple signalling pathway which has elements relevant to the general understanding of signal recognition and transduction across membranes, how signals are relayed between molecules in a pathway, and how adaptation to a persistent signal is achieved. In contrast to many mammalian receptors which signal by oligomerizing upon ligand binding, the chemotaxis receptors are dimeric even in the absence of their ligands, and their signalling does not depend on a monomer-dimer equilibrium. Bacterial chemotaxis receptors are composed of a ligand-binding domain, a transmembrane domain consisting of two helices TM1 and TM2, and a cytoplasmic domain. All known bacterial chemotaxis receptors have a highly conserved cytoplasmic domain, which unites signals from different ligand domains into a single signalling pathway to flagella motors. Here we report the crystal structure of the cytoplasmic domain of a serine chemotaxis receptor of Escherichia coli, which reveals a 200 A-long coiled-coil of two antiparallel helices connected by a 'U-turn'. Two of these domains form a long, supercoiled, four-helical bundle in the cytoplasmic portion of the receptor.     

Three-dimensional structure of CheY, the response regulator of bacterial chemotaxis

Homologies among bacterial signal transduction proteins suggest that a common mechanism mediates processes such as chemotaxis, osmoregulation, sporulation, virulence, and responses to nitrogen, phosphorous and oxygen deprivation. A common kinase-mediated phosphotransfer reaction has recently been identified in chemotaxis, nitrogen regulation, and osmoregulation. In chemotaxis, the CheA kinase passes a phosphoryl group to the cytoplasmic protein CheY, which functions as a phosphorylation-activated switch that interacts with flagellar components to regulate motility. We report here the X-ray crystal structure of the Salmonella typhimurium CheY protein. The determination of the structure was facilitated by the use of site-specific mutagenesis to engineer heavy-atom binding sites. CheY is a single-domain protein composed of a doubly wound five-stranded parallel beta-sheet. The phosphoacceptor site in CheY is probably a cluster of aspartic-acid side chains near the C-terminal edge of the beta-sheet. The pattern of sequence similarity of CheY with components of other regulatory systems can be interpreted in the light of the CheY structure and supports the view that this family of proteins have a common structural motif and active site.     

arrow encodes an LDL-receptor-related protein essential for Wingless signalling

The Wnt family of secreted molecules functions in cell-fate determination and morphogenesis during development in both vertebrates and invertebrates (reviewed in ref. 1). Drosophila Wingless is a founding member of this family, and many components of its signal transduction cascade have been identified, including the Frizzled class of receptor. But the mechanism by which the Wingless signal is received and transduced across the membrane is not completely understood. Here we describe a gene that is necessary for all Wingless signalling events in Drosophila. We show that arrow gene function is essential in cells receiving Wingless input and that it acts upstream of Dishevelled. arrow encodes a single-pass transmembrane protein, indicating that it may be part of a receptor complex with Frizzled class proteins. Arrow is a low-density lipoprotein (LDL)-receptor-related protein (LRP), strikingly homologous to murine and human LRP5 and LRP6. Thus, our data suggests a new and conserved function for this LRP subfamily in Wingless/Wnt signal reception.     

Signal transduction through the CD4 receptor involves the activation of the internal membrane tyrosine-protein kinase p56lck

The CD4 T-cell surface antigen is an integral membrane glycoprotein of relative molecular mass 55,000 which binds class II major histocompatibility complex (MHC) molecules expressed on antigen presenting cells (APCs). It is thought to stabilize physical interactions between T cells and APCs (for a review, see ref. 1). Evidence is accumulating that suggests that CD4 can transduce an independent signal during T-cell activation. It has recently been shown that CD4 expressed on human and murine T cells is physically associated with the Src-related tyrosine protein kinase p56lck (refs 7, 8). These results indicate that CD4 can function as a signal transducer and suggest that tyrosine phosphorylation events may be important in CD4-mediated signalling. Here, we present evidence that cross-linking of the CD4 receptor induces a rapid increase in the tyrosine-specific protein kinase activity of p56lck and is associated with the rapid phosphorylation of one of the subunits (zeta) of the T-cell receptor complex on tyrosine residues. These data provide direct evidence for a specific CD4 signal transduction pathway that is mediated through p56lck and suggest that some of the tyrosine phosphorylation events detected during antigen-mediated T-cell activation may result from signalling through this surface molecule.     

A novel calcium binding site in the galactose-binding protein of bacterial transport and chemotaxis

The refined 1.9-A resolution structure of the periplasmic D-galactose-binding protein (GBP) reveals a calcium ion surrounded by seven ligands, all protein oxygen atoms. A nine-residue loop (amino-acid positions 134-142), which is preceded by a beta-turn and followed by a beta-strand, provides five ligands from every second residue. The last two ligands are supplied by the carboxylate group of Glu 205. The entire GBP Ca2+-binding site adopts a conformation very similar to the site in the 'helix-loop-helix' or 'EF-hand' unit commonly found in intracellular calcium-binding proteins, but without the two helices. Structural analyses have also uncovered the sugar-binding site some 30 A from the calcium and a site for interacting with the membrane-bound trg chemotactic signal transducer approximately 45 A from the calcium. Our results show that a common tight calcium binding site of ancient origin can be tethered to different secondary structures. They also provide the first demonstration of a metal-binding site in a protein which is involved in bacterial active transport and chemotaxis.     

真核生物中支架蛋白家族(IQGAP family)的分子进化分析

支架蛋白家族(IQGAP family)广泛存在于真核生物中,在细胞的信号转导、骨架运动、细胞分裂的过程中都有着重要功能.研究分析IQGAP蛋白家族的分子进化,有助于深入全面地了解整个IQGAP蛋白家族在不同物种中功能.通过使用相关数据库,在35个真核生物物种中检索到了70个支架蛋白家族成员的序列信息,并对其进行了系统的分子进化研究.我们发现:支架蛋白家族基因广泛存在于后生动物(Metazoan)、真菌(Fungi)、变形虫界(Amoebozoa)以及其他一些真核生物中,提示支架蛋白存在于最后的真核生物的共同祖先(Last Eukaryotic Common Ancestor,LECA).系统发育分析表明脊椎动物的IQGAP蛋白和非脊椎动物中代表性IQGAP基因共享一个共同的祖先基因,单个的IQGAP祖先基因在早期脊椎动物中(在四足总纲和硬骨鱼纲的分离之前),发生了两次基因复制事件,导致现存的脊椎动物中3组IQGAP基因(IQGAP1,IQGAP2,IQGAP3)产生.     

Association between GTPase activators for Rho and Ras families.

The ras-related low-molecular-mass GTPases participate in signal transduction involving a variety of cellular functions, including cell-cycle progression, cellular differentiation, cytoskeletal organization, protein transport and secretion. The cycling of these proteins between GTP-bound and GDP-bound states is partially controlled by GTPase activating proteins (GAPs) which stimulate the intrinsic GTP-hydrolysing activity of specific GTPases. The ras GTPase-activating protein (Ras-GAP) forms a complex with a second protein, p190 (M(r) 190,000), in growth-factor stimulated and tyrosine-kinase transformed cells. At its carboxy-terminal end, p190 contains a region that is conserved in the breakpoint cluster region, n-chimaerin, and Rho-GAP. Each of these three proteins exhibits GAP activity for at least one member of the rho family of small GTPases. We have tested recombinant p190 protein for GAP activity on GTPases of the ras, rho and rab families, and show here that p190 can function as a GAP specifically for members of the rho family. Consequently, the formation of a complex between Ras-GAP and p190 in growth-factor stimulated cells may allow the coupling of signalling pathways that involve ras and rho GTPases.     

Different beta-subunits determine G-protein interaction with transmembrane receptors.

Regulatory GTP-binding proteins (G proteins) are membrane-attached heterotrimers (alpha, beta, gamma) that mediate cellular responses to a wide variety of extracellular stimuli. They undergo a cycle of guanine-nucleotide exchange and GTP hydrolysis, during which they dissociate into alpha-subunit and beta gamma complex. The roles of G-protein alpha-subunits in these processes and for the specificity of signal transduction are largely established; the beta- and gamma-subunits are essential for receptor-induced G-protein activation and seem to be less diverse and less specific. Although the complementary DNAs for several beta-subunits have been cloned, isolated subunits have only been studied as beta gamma complexes. Functional differences have been ascribed to the gamma-subunit on the basis of extensive sequence similarity among beta-subunits and apparent heterogeneity in gamma-subunit sequences. Beta gamma complexes can interact directly or indirectly with different effectors. They seem to be interchangeable in their interaction with pertussis toxin-sensitive alpha-subunits, so we tested this by microinjecting antisense oligonucleotides into nuclei of a rat pituitary cell line to suppress the synthesis of individual beta-subunits selectively. Here we show that two out of four subtypes of beta-subunits tested (beta 1 and beta 3) are selectively involved in the signal transduction cascades from muscarinic M4 (ref. 4) and somatostatin receptors, respectively, to voltage-dependent Ca2+ channels.     

植物中的DREB类转录因子

DREB转录因子属于AP2/EREBP转录因子家族.AP2/EREBP转录因子是特异存在于植物中,与植物发育密切相关的一类转录因子的总称.该家族有五个亚家族,其家族成员的蛋白质序列均含有保守的AP2 domain.其中,DREB转录因子特异地与DRE顺式作用元件相结合,在调节低温,干旱和高盐等胁迫反应时具有重要作用.一个DREB转录因子可以调控下游的多个抗逆基因,从而使植物产生抗逆性.植物抗逆反应是一个非常复杂的过程,目前人们对其信号转导正在进行进一步的探究.     

Stimulation of specific GTP binding and hydrolysis activities in lymphocyte membrane by interleukin-2

Interleukin-2 (IL-2) is a polypeptide growth factor which stimulates the proliferation and differentiation of T lymphocytes. The receptor for IL-2 is expressed on activated T lymphocytes, cloned IL-2 dependent cells and several other cell types. Analysis of the primary structure and of immune-precipitated receptor suggests that this molecule has no intrinsic signal transduction function, unlike other growth factors. IL-2 interaction with a high affinity receptor has been shown, however, to activate the calcium/phospholipid-dependent protein kinase C (PK-C) presumably via phosphoinositide hydrolysis. Members of a family of closely related guanine nucleotide binding proteins (G proteins) regulate a diverse group of metabolic events. Two of them, Gs and Gi, stimulate and inhibit adenylate cyclase activity respectively, and other G proteins are involved in diverse signal transduction system. Another member, Go, has no known function and activation of phospholipase C has been attributed to the action of an unidentified G protein, Gp. Since it has been observed that IL-2 inhibits the catalytic activity of adenylate cyclase and that agents such as PGE2 which stimulate adenylate cyclase activity inhibit the lymphoproliferative response to IL-2, association of GTP binding proteins with IL-2 signal transduction was investigated. In this report we describe for the first time the participation of a GTP binding protein in the action of a polypeptide growth factor, interleukin-2.     

Prediction and systematic study of protein-protein interaction networks of Leptospira interrogans

Leptospirosis, a serious and contagious zoonotic bacterial disease that havocs livers and kidneys in hu-mans and some animals, occurs in both urban and rural environments worldwide. It is caused by the genus lep- tospira, a corkscrew-shaped bacterium[1]. Though the genomes of Leptospira interrogans serovars Lai strain lai[2] and Leptospira interrogans serovars Copenhageni strain L1-130[3] were recently sequenced and many molecular and cellular studies on leptospires have been conducted, the …     

E3 ubiquitin ligase that recognizes sugar chains

N-glycosylation of proteins in the endoplasmic reticulum (ER) has a central role in protein quality control. Here we report that N-glycan serves as a signal for degradation by the Skp1-Cullin1-Fbx2-Roc1 (SCF(Fbx2)) ubiquitin ligase complex. The F-box protein Fbx2 (ref. 4) binds specifically to proteins attached to N-linked high-mannose oligosaccharides and subsequently contributes to ubiquitination of N-glycosylated proteins. Pre-integrin beta 1 is a target of Fbx2; these two proteins interact in the cytosol after inhibition of the proteasome. In addition, expression of the mutant Fbx2 Delta F, which lacks the F-box domain that is essential for forming the SCF complex, appreciably blocks degradation of typical substrates of the ER-associated degradation pathway. Our results indicate that SCF(Fbx2) ubiquitinates N-glycosylated proteins that are translocated from the ER to the cytosol by the quality control mechanism.     

Crystal structure of metarhodopsin II

G-protein-coupled receptors (GPCRs) are seven transmembrane helix (TM) proteins that transduce signals into living cells by binding extracellular ligands and coupling to intracellular heterotrimeric G proteins (Gαβγ). The photoreceptor rhodopsin couples to transducin and bears its ligand 11-cis-retinal covalently bound via a protonated Schiff base to the opsin apoprotein. Absorption of a photon causes retinal cis/trans isomerization and generates the agonist all-trans-retinal in situ. After early photoproducts, the active G-protein-binding intermediate metarhodopsin II (Meta?II) is formed, in which the retinal Schiff base is still intact but deprotonated. Dissociation of the proton from the Schiff base breaks a major constraint in the protein and enables further activating steps, including an outward tilt of TM6 and formation of a large cytoplasmic crevice for uptake of the interacting C terminus of the Gα subunit. Owing to Schiff base hydrolysis, Meta?II is short-lived and notoriously difficult to crystallize. We therefore soaked opsin crystals with all-trans-retinal to form Meta?II, presuming that the crystal's high concentration of opsin in an active conformation (Ops*) may facilitate all-trans-retinal uptake and Schiff base formation. Here we present the 3.0?? and 2.85?? crystal structures, respectively, of Meta?II alone or in complex with an 11-amino-acid C-terminal fragment derived from Gα (GαCT2). GαCT2 binds in a large crevice at the cytoplasmic side, akin to the binding of a similar Gα-derived peptide to Ops* (ref. 7). In the Meta?II structures, the electron density from the retinal ligand seamlessly continues into the Lys?296 side chain, reflecting proper formation of the Schiff base linkage. The retinal is in a relaxed conformation and almost undistorted compared with pure crystalline all-trans-retinal. By comparison with early photoproducts we propose how retinal translocation and rotation induce the gross conformational changes characteristic for Meta?II. The structures can now serve as models for the large GPCR family.     

Tyrosine-containing motif that transduces cell activation signals also determines internalization and antigen presentation via type III receptors for IgG.

Type III receptors for IgG (Fc gamma RII; ref. 1), high-affinity IgE receptors (Fc epsilon RI; ref. 2), as well as the T- and B-cell antigen receptors, consist of multiple components with specialized ligand-binding and signal transduction functions. Fc gamma RII alpha (ligand-binding) and gamma (signal-transducing) subunits are expressed in macrophages, a cell type involved in the uptake of antigen, its processing and the presentation of the resulting peptides to major histocompatibility complex class II-restricted T lymphocytes. Here we show that murine Fc gamma RIII, transfected into Fc gamma R-negative antigen-presenting B-lymphoma cells, mediate rapid ligand internalization and strongly increase the efficiency of antigen presentation when antigen is complexed to IgG. Efficient internalization and antigen presentation via Fc gamma RIII did not require the cytoplasmic domain of the ligand-binding alpha-chain, but did require the gamma-subunit. Using chimaeric molecules, we show that gamma-chain contains a signal for receptor internalization and that the mutation of either of the two tyrosine residues present in its cytoplasmic domain prevents efficient internalization and antigen presentation of immune complexes. Thus, associated chains and their tyrosine-containing motif are not exclusively involved in cell activation, but also determine multimeric receptor internalization.     

SOCS-2和SOCS-3通过IGF-1和GH信号转导系统作用于成肌细胞的分化

新近发现的细胞因子信号转导抑制因子（SOCS）家族，因其能够通过Janus激酶-信号传导和转录激活子（JAK-STAT）信号传导通路来反馈调节生长因子的信号或者抑制细胞因子的信号转导而倍受研究人员重视。一些研究表明，SOCS-3在促进成肌细胞分化和抑制白介素6（IL-6）导致的细胞炎症过程中具有重要的作用。综合大量关于细胞因子信号转导抑制因子家族的文献报道，文章分析了近几年SOCS-2和SOCS-3与IGF-1和GH信号转导关系的研究，特别是关于SOCS-3在成肌细胞分化过程中的研究，认为可以将SOCS-2和SOCS-3作为细胞内生长信号调节和促进动物肌肉发育的潜在因子进行研究。     

A chloride channel widely expressed in epithelial and non-epithelial cells.

Chloride channels have several functions, including the regulation of cell volume, stabilizing membrane potential, signal transduction and transepithelial transport. The plasma membrane Cl- channels already cloned belong to different structural classes: ligand-gated channels, voltage-gated channels, and possibly transporters of the ATP-binding-cassette type (if the cystic fibrosis transmembrane regulator is a Cl- channel). The importance of chloride channels is illustrated by the phenotypes that can result from their malfunction: cystic fibrosis, in which transepithelial transport is impaired, and myotonia, in which ClC-1, the principal skeletal muscle Cl- channel, is defective. Here we report the properties of ClC-2, a new member of the voltage-gated Cl- channel family. Its sequence is approximately 50% identical to either the Torpedo electroplax Cl- channel, ClC-0 (ref. 8), or the rat muscle Cl- channel, ClC-1 (ref. 9). Isolated initially from rat heart and brain, it is also expressed in pancreas, lung and liver, for example, and in pure cell lines of fibroblastic, neuronal, and epithelial origin, including tissues and cells affected by cystic fibrosis. Expression in Xenopus oocytes induces Cl- currents that activate slowly upon hyperpolarization and display a linear instantaneous current-voltage relationship. The conductivity sequence is Cl- greater than or equal to Br- greater than I-. The presence of ClC-2 in such different cell types contrasts with the highly specialized expression of ClC-1 (ref. 9) and also with the cloned cation channels, and suggests that its function is important for most cells.