Adenosine receptors interacting proteins (ARIPs): Behind the biology of adenosine signaling

Adenosine is a well known neuromodulator in the central nervous system. As a consequence, adenosine can be beneficial in certain disorders and adenosine receptors will be potential targets for therapy in a variety of diseases. Adenosine receptors are G protein-coupled receptors, and are also expressed in a large variety of cells and tissues. Using these receptors as a paradigm of G protein-coupled receptors, the present review focus on how protein-protein interactions might contribute to neurotransmitter/neuromodulator regulation, based on the fact that accessory proteins impinge on the receptor/G protein interaction and therefore modulate receptor functioning. Besides affecting receptor signaling, these accessory components also play a key role in receptor trafficking, internalization and desensitization, as it will be reviewed here. In conclusion, the finding of an increasing number of adenosine receptors interacting proteins, and specially the molecular and functional integration of these accessory proteins into receptorsomes, will open new perspectives in the understanding of particular disorders where these receptors have been proved to be involved.     

Phospholipid binding by proteins of the spectrin family: a comparative study

Erythroid and neuronal spectrin (fodrin) are both known to interact strongly with the aminophospholipids that occur in the inner leaflet of plasma membranes. In erythroid spectrin the positions of the binding sites within the constituent (alphaI and betaI) polypeptide chains have been defined, and also the importance of the lipid interaction in regulating the properties of the membrane. Here we report the locations of the corresponding binding sites in the alphaII and betaII chains that make up the fodrin molecule. Of the 10 lipid-binding repeats in the erythroid spectrin chains 5 are conserved in fodrin; one cluster of 3 consecutive structural repeating units in alphaI erythroid spectrin (repeats 8-10) is displaced by one repeat in alphaII fodrin (repeats 9-11). Fodrin also contains one binding site at the N-terminus of the alphaII chain, not present in the erythroid protein. The regions of the two spectrins containing equivalent lipid-binding sites show a much higher degree of sequence identity than corresponding repeats that do not share this property. The evolutionary conservation of the distribution of a large proportion of strong lipid-binding sites in the polypeptide chains of these two proteins of disparate character argues for a specific function of fodrin-phospholipid interactions in the neuron.     

应用酵母双杂交技术寻找与GGNBP相互作用的蛋白质

生殖细胞缺陷症(gcd)小鼠突变体是20个世纪90年代初发现的一种不育突变小鼠,其不育原因是由于其胚胎期原始生殖细胞的数目低于正常。FancL(也叫Pog)的缺失是引起gcd突变小鼠的原因,FancL基因缺失后可能影响了小鼠胚胎期原始生殖细胞的增殖／存活和成年期小鼠精母细胞的减数分裂。FANCL是一种含有PHD结构域的泛素E3连接酶,是Fanconi贫血复合物的组分之一。在生殖细胞中,FANCL与GGN1和GGN3相互作用,GGN1和GGN2又与一种新的蛋白质GGNBP特异作用。但GGNBP蛋白的功能还不清楚。为了研究GGNBP的功能以及揭示更多的参与该过程的蛋白质,运用Clontech公司新开发的第3套酵母双杂交系统,以GGNBP为诱饵从成年小鼠睾丸cDNA库中筛选与其相互作用的蛋白质基因,发现了一个主要在睾丸中表达的新的基因,其编码的蛋白质产物在酵母系统中与GGNBP特异作用。     

RRM proteins interacting with the cap region of topoisomerase I

RNA recognition motif (RRM) domains bind both nucleic acids and proteins. Several proteins that contain two closely spaced RRM domains were previously found in protein complexes formed by the cap region of human topoisomerase I, a nuclear enzyme responsible for DNA relaxation or phosphorylation of SR splicing proteins. To obtain molecular insight into specific interactions between the RRM proteins and the cap region of topo I we examined their binary interactions using the yeast two-hybrid system. The interactions were established for hnRNP A1, p54(nrb) and SF2/ASF, but not for hnRNP L or HuR. To identify the amino acid pattern responsible for binding, experimental mutagenesis was employed and computational modelling of these processes was carried out. These studies revealed that two RRM domains and six residues of the consensus sequence are required for the binding to the cap region. On the basis of the above data, a structural model for the hnRNP A1-topoisomerase I complex was proposed. The main component of the hnRNP A1 binding site is a hydrophobic pocket on the beta-surface of the first RRM domain, similar to that described for Y14 protein interacting with Mago. We demonstrated that the interaction between RRM domains and the cap region was important for the kinase reaction catalyzed by topoisomerase I. Together with the previously described inhibitory effect of RRM domains of SF2/ASF on DNA cleavage, the above suggests that the binding of RRM proteins could regulate the activity of topoisomerase I.     

G蛋白信号调节因子的结构分类和功能

G蛋白信号调节因子是能够直接与激活的Gα亚基结合,显著刺激Gα亚基上的GTP酶活性,加速GTP水解,从而灭活或终止G蛋白信号的一组分子大小各异的多功能蛋白质家族。它们都共同拥有一个130个氨基酸的保守的RGS结构域,其功能是结合激活的Gα亚基,负调节G蛋白信号。许多RGS蛋白还拥有非RGS结构域,能够结合其它信号蛋白,从而整合和调节G蛋白信号之间以及G蛋白和其它信号系统之间的关系。     

The effect of endogenous proteases on the spectrin binding proteins of human erythrocytes

We have demonstrated that in human erythrocyte ghosts endogenous proteolytic activity is responsible for the digestion of the spectrin binding proteins (bands 2.1 to 2.6). The pH optimum, cofactor requirements and inhibitor sensitivity have been established. Our results indicate that proteolysis of bands 2.1 to 2.6 and the formation of 3′, a fragment containing an active spectrin binding site, can occur through two enzymatic pathways: a cascade of consecutive proteolytic cleavages of the spectrin binding proteins inhibited by phenylmethylsulfonyl fluoride or a Ca²⁺-stimulated, phenylmethylsulfonyl fluoride-insensitive, EDTA-inhibited cleavage of band 2.1 to band 2.3, followed by digestion to band 3′ by phenylmethylsulfonyl fluoride-inhibitable enzymes. These findings may provide the techniques necessary to prevent proteolysis of the spectrin binding proteins during purification and reconstitution experiments and provide insight into how they are formed in vivo.     

Association of spectrin with its membrane attachment site restricts lateral mobility of human erythrocyte integral membrane proteins

Interactions between spectrin and the inner surface of the human erythrocyte membrane have been implicated in the control of lateral mobility of the integral membrane proteins. We report here that incubation of “leaky” erythrocytes with a water-soluble proteolytic fragment containing the membrane attachment site for spectrin achieves a selective and controlled dissociation of spectrin from the membrane, and increases the rate of lateral mobility of fluorescein isothiocyanate-labeled integral membrane proteins (> 70% of label in band 3 and PAS-1). Mobility of membrane proteins is measured as an increase in the percentage of uniformly fluorescent cells with time after fusion of fluorescent with nonfluorescent erythrocytes by Sendai virus. The cells are permeable to macromolecules since virus-fused erythrocytes lose most of their hemoglobin. The membrane attachment site for spectrin has been solubilized by limited proteolysis of inside-out erythrocyte vesicles and has been purified (V). Bennett, J Biol Chem 253:2292 (1978). This 72,000-dalton fragment binds to spectrin in solution, competitively inhibits association of ³²P-spectrin with inside-out vesicles with a K_i of 10^?7M, and causes rapid dissociation of ³²P-spectrin from vesicles. Both acid-treated 72,000-dalton fragment and the 45,000 dalton-cytoplasmic portion of band 3, which also was isolated from the proteolytic digest, have no effect on spectrin binding, release, or membrane protein mobility. The enhancement of membrane protein lateral mobility by the same polypeptide that inhibits binding of spectrin to inverted vesicles and displaces spectrin from these vesicles provides direct evidence that the interaction of spectrin with protein components in the membrane restricts the lateral mobility of integral membrane proteins in the erythrocyte.     

Exploiting the co-evolution of interacting proteins to discover interaction specificity

Protein interactions are fundamental to the functioning of cells, and high throughput experimental and computational strategies are sought to map interactions. Predicting interaction specificity, such as matching members of a ligand family to specific members of a receptor family, is largely an unsolved problem. Here we show that by using evolutionary relationships within such families, it is possible to predict their physical interaction specificities. We introduce the computational method of matrix alignment for finding the optimal alignment between protein family similarity matrices. A second method, 3D embedding, allows visualization of interacting partners via spatial representation of the protein families. These methods essentially align phylogenetic trees of interacting protein families to define specific interaction partners. Prediction accuracy depends strongly on phylogenetic tree complexity, as measured with information theoretic methods. These results, along with simulations of protein evolution, suggest a model for the evolution of interacting protein families in which interaction partners are duplicated in coupled processes. Using these methods, it is possible to successfully find protein interaction specificities, as demonstrated for >18 protein families.     

The structure of the plakin domain of plectin reveals a non-canonical SH3 domain interacting with its fourth spectrin repeat

Plectin belongs to the plakin family of cytoskeletal crosslinkers, which is part of the spectrin superfamily. Plakins contain an N-terminal conserved region, the plakin domain, which is formed by an array of spectrin repeats (SR) and a Src-homology 3 (SH3), and harbors binding sites for junctional proteins. We have combined x-ray crystallography and small angle x-ray scattering (SAXS) to elucidate the structure of the central region of the plakin domain of plectin, which corresponds to the SR3, SR4, SR5, and SH3 domains. The crystal structures of the SR3-SR4 and SR4-SR5-SH3 fragments were determined to 2.2 and 2.95 Å resolution, respectively. The SH3 of plectin presents major alterations as compared with canonical Pro-rich binding SH3 domains, suggesting that plectin does not recognize Pro-rich motifs. In addition, the SH3 binding site is partially occluded by an intramolecular contact with the SR4. Residues of this pseudo-binding site and the SR4/SH3 interface are conserved within the plakin family, suggesting that the structure of this part of the plectin molecule is similar to that of other plakins. We have created a model for the SR3-SR4-SR5-SH3 region, which agrees well with SAXS data in solution. The three SRs form a semi-flexible rod that is not altered by the presence of the SH3 domain, and it is similar to those found in spectrins. The flexibility of the plakin domain, in analogy with spectrins, might contribute to the role of plakins in maintaining the stability of tissues subject to mechanical stress.     

Direct association of tristetraprolin with the nucleoporin CAN/Nup214

Tristetraprolin (TTP) is a widely expressed, zinc finger-containing protein that has been implicated in the regulation of TNFalpha production in mice. Stimulus-dependent cytoplasmic translocation of TTP has been demonstrated in several cells. In this report we used the yeast two-hybrid screen to identify proteins able to interact with full length, human TTP. One of the isolated TTP-interacting clones encoded the FG repeat region of the nuclear pore protein Nup214. Full length Nup214 co-precipitated with TTP from resting and LPS-stimulated THP-1 cells, indicating that this interaction occurred in intact cells. The ability of TTP to associate with Nup214 was dependent on two intact zinc fingers within TTP. In contrast to wild type TTP that localized primarily in the cytosol, a mutant unable to associate with Nup214 localized throughout the cell, suggesting that the interaction with Nup214 regulates TTP localization.     

The 22.5 kDa spectrin-binding domain of ankyrinR binds spectrin with high affinity and changes the spectrin distribution in cells in vivo

It was previously shown that ankyrins play a crucial role in the membrane skeleton arrangement. Purifying ankyrinR obtained from erythrocytes is a time-consuming process. Therefore, cloned and bacterially expressed ankyrinR–spectrin-binding domain (AnkSBD) is a demanded tool for studying spectrin–ankyrin interactions. In this communication, we report on the cloning and purification of AnkSBD and describe the results of binding experiments, in which we showed high-affinity interactions between the AnkSBD construct and isolated erythrocyte or non-erythroid spectrins. pEGFP–AnkSBD-transfected cells co-localised with non-erythroid spectrin in HeLa cells. The functional interactions of the AnkSBD construct in vivo and in vitro open many possibilities to study the structure and function of this domain, which has not yet been as extensively studied when compared to the aminoterminal domain of this protein.     

Biochemical analysis of the interactions of IQGAP1 C-terminal domain with CDC42

AIM:To understand the interaction of human IQGAP1 and CDC42,especially the effects of phosphorylation and a cancer-associated mutation. METHODS:Recombinant CDC42 and a novel C-termi- nal fragment of IQGAP1 were expressed in,and puri- fied from,Escherichia coli.Site directed mutagenesis was used to create coding sequences for three phos- phomimicking variants(S1441E,S1443D and S1441E/ S1443D)and to recapitulate a cancer-associated mu- tation(M1231I).These variant proteins were also ex- pressed and purified.Protein-protein crosslinking using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide was used to investigate interactions between the C-terminal fragment and CDC42.These interactions were quanti- fied using surface plasmon resonance measurements.Molecular modelling was employed to make predictions about changes to the structure and flexibility of the protein which occur in the cancer-associated variant. RESULTS:The novel,C-terminal region of human IQGAP1 (residues 877-1558)is soluble following expression and purification.It is also capable of binding to CDC42,as judged by crosslinking experiments.Interaction appears to be strongest in the presence of added GTP.The three phosphomimicking mutants had different affini- ties for CDC42.S1441E had an approximately 200-fold reduction in affinity compared to wild type.This was caused largely by a dramatic reduction in the associa- tion rate constant.In contrast,both S1443D and the double variant S1441E/S1443D had similar affinities to the wild type.The cancer-associated variant,M1231I, also had a similar affinity to wild type.However,in the case of this variant,both the association and dis- sociation rate constants were reduced approximately 10-fold.Molecular modelling of the M1231I variant, based on the published crystal structure of part of the C-terminal region,revealed no gross structural changes compared to wild type(root mean square deviation of 0.564over 5556 equivalent atoms).However,pre- dictions of the flexibility of the polypeptide backbone suggested that some regions of the variant protein had greatly increased rigidity compared to wild type.One such region is a loop linking the proposed CDC42 bind- ing site with the helix containing the altered residue.It is suggested that this increase in rigidity is responsible for the observed changes in association and dissocia- tion rate constants. CONCLUSION:The consequences of introducing nega- tive charge at Ser-1441 or Ser-1443 in IQGAP1 are dif- ferent.The cancer-associated variant M1231I exerts its effects partly by rigidifying the protein.     

Zinc(II)cyclen-peptide conjugates interacting with the weak effector binding state of Ras

Zinc(II)cyclen-peptide hybrid compounds and bis-zinc(II)cyclen complexes are prepared as potential binders of the guanine nucleotide binding protein Ras, an important molecular switch in cellular signal transduction. The design of the compounds is based on the previous observation that zinc(II)cyclen complexes could serve as lead compounds for inhibitors of Ras-effector interaction and thus be able to interrupt Ras induced signal transduction. Zinc(II)cyclen selectively stabilizes conformational state 1 of active Ras, a conformational state with drastically decreased affinity to effector proteins like Raf-kinase. To achieve higher binding affinities of such Ras-Raf interaction inhibitors, zinc(II)cyclen conjugates with short peptides, derived from the sequence of the Ras-activator SOS, were prepared by solid phase synthesis protocols. Dinuclear bis-zinc(II)cyclen complexes were obtained from alkyne-azide cycloaddition reactions. NMR investigations of the prepared compounds revealed that the peptide conjugates do not lead to an increase in Ras binding affinity of the metal complex-peptide conjugates. The dinuclear zinc complexes lead to an immediate precipitation of the protein prohibiting spectroscopic investigations of their binding.     

DAZAP1 interacts via its RNA-recognition motifs with the C-termini of other RNA-binding proteins

The turnover and translation of many human mRNAs is regulated by AU-rich elements present in their 3′untranslated region, which bind various trans acting factors. We previously identified a trans acting factor that interacts with these cis elements as DAZAP1 (deleted in Azoospermia (DAZ)-Associated Protein 1), whose interaction with the germ cell-specific protein DAZ was disrupted by the phosphorylation of DAZAP1. Here we have identified several other RNA-binding proteins as binding partners for DAZAP1 in non-germinal cells. Unlike DAZ, these interactions occur between the RNA recognition motifs of DAZAP1 and the C-termini of the binding partners and in a phosphorylation-independent manner. The results suggest that DAZAP1 is a component of complexes that are crucial for the degradation and silencing of mRNA.     

Alterations in human erythrocyte shape and the state of spectrin and phospholipid phosphorylation induced by cholesterol depletion

Cholesterol depletion of erythrocytes, obtained after incubation with phosphatidylcholine vesicles, induces in most of the experiments: (1) a discocytestomatocyte transformation as observed by scanning electron microscopy; (2) a specific decrease in spectrin phosphorylation of intact erythrocytes; (3) an increase in lipid phosphorylation. It is concluded that the effect of cholesterol on erythrocyte shape is probably mediated through its action on the activity o of membrane-bound enzymes, proteases or kinases.     

A microtiter plate assay for assessing the interaction of eukaryotic initiation factor eIF4E with eIF4G and eIF4E binding protein-1

Modulation of interactions among proteins is an important mechanism for regulating both the subcellular location and the function of proteins. An example of the importance of protein-protein interaction is the reversible association of eukaryotic initiation factor eIF4E with the eIF4E binding proteins 4E-BP1 and eIF4G. When bound to 4E-BP1, eIF4E cannot bind to eIF4G to form the active eIF4F complex, an event that is required for the binding of mRNA to the ribosome. Thus, association of eIF4E with 4E-BP1 represses mRNA translation by preventing the binding of mRNA to the ribosome. Previous studies have measured the amount of 4E-BP1 or eIF4G bound to eIF4E by either affinity chromatography or immunoprecipitation of eIF4E followed by Western blot analysis for quantitation of 4E-BP1 and eIF4G. Both of these techniques have significant limitations. In the present study, we describe a microtiter plate-based assay for quantitation of the amount of 4E-BP1 and eIF4G bound to eIF4E that obviates many of the limitations of the earlier approaches. It also has the advantage that absolute amounts of the individual proteins can be easily estimated. The approach should be applicable to the study of a wide variety of protein-protein interactions.