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PubMed2005

Constitutive formation of oligomeric complexes between family B G protein-coupled vasoactive intestinal polypeptide and secretin receptors.

Formation of oligomeric complexes of family A G protein-coupled receptors has been shown to influence their function and regulation. However, little is known about the existence of such complexes for family B receptors in this superfamily. We previously used bioluminescence resonance energy transfer (BRET) to demonstrate that the prototypic family B secretin receptor forms ligand-independent oligomeric complexes. Here, we show that subtypes of human vasoactive intestinal polypeptide receptors (VPAC1 and VPAC2) that represent the closest structurally related receptors to the secretin receptor also form constitutive oligomers with themselves and with the secretin receptor. We prepared tagged constructs expressing Renilla reniformis luciferase, yellow fluorescent protein, or cyan fluorescent protein at the carboxyl terminus of VPAC1, VPAC2, and secretin receptors, and performed BRET and morphologic fluorescence resonance energy transfer (FRET) studies with all combinations. The specificity of the BRET and FRET signals was confirmed by control studies. These constructs bound their natural ligands specifically and saturably, with these agonists able to elicit full cAMP responses. BRET studies showed that, like the secretin receptor, both VPAC receptors exhibited constitutive homo-oligomerization in COS cells. Unlike secretin receptor oligomers that were unaffected by ligand binding, the VPAC receptor homo-oligomers were modulated by vasoactive intestinal polypeptide. In addition, each of these three receptors formed hetero-oligomers with each other. The VPAC1-VPAC2 hetero-oligomers were modulated by vasoactive intestinal polypeptide binding, whereas the secretin-VPAC1 and secretin-VPAC2 receptor hetero-oligomers were unaffected by ligand treatment. Morphologic FRET studies demonstrated that each of the homo-oligomers and the VPAC1-VPAC2 receptor hetero-oligomers reached the cell surface, where receptor interactions were clear. However, coexpression of secretin receptors with either type of VPAC receptor resulted in intracellular trapping of the hetero-oligomeric complexes within the biosynthetic pathway. These studies provide new insight into the ability of family B G protein-coupled receptors to associate with each other in cells.

Animals↗
PubMed1998

Essential role of CED-4 oligomerization in CED-3 activation and apoptosis.

Control of the activation of apoptosis is important both in development and in protection against cancer. In the classic genetic model Caenorhabditis elegans, the pro-apoptotic protein CED-4 activates the CED-3 caspase and is inhibited by the Bcl-2-like protein CED-9. Both processes are mediated by protein-protein interaction. Facilitating the proximity of CED-3 zymogen molecules was found to induce caspase activation and cell death. CED-4 protein oligomerized in cells and in vitro. This oligomerization induced CED-3 proximity and competed with CED-4:CED-9 interaction. Mutations that abolished CED-4 oligomerization inactivated its ability to activate CED-3. Thus, the mechanism of control is that CED-3 in CED-3:CED-4 complexes is activated by CED-4 oligomerization, which is inhibited by binding of CED-9 to CED-4.

Apoptosis↗
PubMed2001

The alpha-helix 4 residue, Asn135, is involved in the oligomerization of Cry1Ac1 and Cry1Ab5 Bacillus thuringiensis toxins.

The insecticidal Cry toxins produced by the bacterium Bacillus thuringiensis are comprised of three structural domains. Domain I, a seven-helix bundle, is thought to penetrate the insect epithelial cell plasma membrane through a hairpin composed of alpha-helices 4 and 5, followed by the oligomerization of four hairpin monomers. The alpha-helix 4 has been proposed to line the lumen of the pore, whereas some residues in alpha-helix 5 have been shown to be responsible for oligomerization. Mutation of the Cry1Ac1 alpha-helix 4 amino acid Asn135 to Gln resulted in the loss of toxicity to Manduca sexta, yet binding was still observed. In this study, the equivalent mutation was made in the Cry1Ab5 toxin, and the properties of both wild-type and mutant toxin counterparts were analyzed. Both mutants appeared to bind to M. sexta membrane vesicles, but they were not able to form pores. The ability of both N135Q mutants to oligomerize was also disrupted, providing the first evidence that a residue in alpha-helix 4 can contribute to toxin oligomerization.

Animals↗
PubMed2000

The multifunctional bacteriophage P2 cox protein requires oligomerization for biological activity.

The Cox protein of bacteriophage P2 is a multifunctional protein of 91 amino acids. It is directly involved in the site-specific recombination event leading to excision of P2 DNA out of the host chromosome. In this context, it functions as an architectural protein in the formation of the excisome. Cox is also a transcriptional repressor of the P2 Pc promoter, thereby ensuring lytic growth. Finally it promotes derepression of prophage P4, a nonrelated defective satellite phage, by activating the P4 P(LL) promoter that controls P4 DNA replication. In this case it binds upstream of the P(LL) promoter, which normally is activated by the P4 Delta protein. In this work we have analyzed the native form of the Cox protein in vivo, using a bacteriophage lambda cI-based oligomerization assay system, and in vitro, using gel filtration, cross-linking agents, and gel retardation assays. We found that P2 Cox has a strong oligomerization function in vivo as well as in vitro. The in vitro analysis indicates that its native form is a tetramer that can self-associate to octamers. Furthermore we show that oligomerization is necessary for the biological activity by characterizing different cox mutants and that oligomerization is mediated by the C-terminal region.

Amino Acid Sequence↗
PubMed1994

Role of oligomerization of the S13 Env-Sea oncoprotein in cell transformation.

The env-sea oncogene is a fusion of the S13 viral envelope gene, env, and cell-derived sequences encoding a tyrosine kinase domain, termed sea. The Env-Sea oncoprotein is synthesized as a precursor of 155 kDa which undergoes proteolytic processing to generate a disulfide-linked complex of the proteins gp85 and gp70. We analyzed the oligomeric state of the Env-Sea oncoprotein in S13-transformed cells and demonstrate that both gp155 and the gp85-gp70 complex can oligomerize. To address the relevance of these oligomers in transformation by S13, a mutant that is temperature sensitive for the transformed phenotype was used. The tyrosine-phosphorylated oligomers of gp155 were found at the nonpermissive temperature, and thus oligomerization per se appears to be insufficient to elicit a transformed phenotype. Efficient intracellular transport of gp155 appears to be required to generate a tyrosine-phosphorylated oligomer of the gp85-gp70 complex, the presence of which correlates with the transformed phenotype. This gp85-gp70 complex appeared to have a higher level of kinase activity than the other forms of the Env-Sea protein. These results suggest that oligomerization, transport, and intracellular localization represent levels at which the oncogenic activity of the Env-Sea oncoprotein may be regulated.

Alpharetrovirus↗
PubMed1997

Epitope map of human immunodeficiency virus type 1 gp41 derived from 47 monoclonal antibodies produced by immunization with oligomeric envelope protein.

The biologically relevant form of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein is oligomeric, with the major points of contact between oligomeric partners located in the ectodomain of gp41. To identify and map conserved epitopes and regions in gp41 where structure is influenced by quaternary interactions, we used a panel of 38 conformation-dependent and 9 conformation-independent anti-gp41 monoclonal antibodies (MAbs) produced by immunization of mice with oligomeric Env protein. By cross-competition experiments using these MAbs and several others previously described, six distinct antigenic determinants were identified and mapped. Three of these determinants are conformational in nature and dependent in part on Env oligomeric structure. MAbs to two of these determinants were broadly cross-reactive with Env proteins derived from primary virus strains. The prevalence of antibodies in HIV-1-positive human sera to the antigenic determinants was determined by the ability of such sera to block binding of MAbs to Env protein. Strong blocking activity that correlated with cross-reactivity was found.

Amino Acid Sequence↗
PubMed1997

Human immunodeficiency virus type 1 and 2 envelope glycoproteins oligomerize through conserved sequences.

Hetero-oligomerization between human immunodeficiency virus type 2 (HIV-2) envelope glycoprotein (Env) truncation mutants and epitope-tagged gp160 is dependent on the presence of gp41 transmembrane protein (TM) amino acids 552 to 589, a putative amphipathic alpha-helical sequence. HIV-2 Env truncation mutants containing this sequence were also able to form cross-type hetero-oligomers with HIV-1 Env. HIV-2/HIV-1 hetero-oligomerization was, however, more sensitive to disruption by mutagenesis or increased temperature. The conservation of the Env oligomerization function of the HIV-1 and HIV-2 alpha-helical sequences suggests that retroviral TM alpha-helical motifs may have a universal role in oligomerization.

Amino Acid Sequence↗
PubMed2002

Oligomeric structure of the human immunodeficiency virus type 1 envelope protein on the virion surface.

The envelope protein (Env) of human immunodeficiency virus type 1 forms homo-oligomers in the endoplasmic reticulum. The oligomeric structure of Env is maintained after cleavage in a Golgi compartment and transport to the surfaces of infected cells, where incorporation into budding virions takes place. Here, we use biophysical techniques to assess the oligomeric valency of virion-associated Env prior to fusion activation. Virion-associated Env oligomers were stabilized by chemical cross-linking prior to detergent extraction and were purified by immunoaffinity chromatography. Gel filtration revealed a single predominant oligomeric species, and sedimentation equilibrium analysis-derived mass values indicated a trimeric structure. Determination of the masses of individual Env molecules by scanning transmission electron microscopy demonstrated that virion-associated Env was trimeric, and a triangular morphology was observed in 20 to 30% of the molecules. These results, which firmly establish the oligomeric structure of human immunodeficiency virus virion-associated Env, parallel those of our previous analysis of the simian immunodeficiency virus Env.

Biopolymers↗
PubMed2004

Oligomerization of Hantavirus N protein: C-terminal alpha-helices interact to form a shared hydrophobic space.

The structure of the nucleocapsid protein of bunyaviruses has not been defined. Earlier we have shown that Tula hantavirus N protein oligomerization is dependent on the C-terminal domains. Of them, the helix-loop-helix motif was found to be an essential structure. Computer modeling predicted that oligomerization occurs via helix protrusions, and the shared hydrophobic space formed by amino acids residues 380-IILLF-384 in the first helix and 413-LI-414 in the second helix is responsible for stabilizing the interaction. The model was validated by two approaches. First, analysis of the oligomerization capacity of the N protein mutants performed with the mammalian two-hybrid system showed that both preservation of the helix structure and formation of the shared hydrophobic space are crucial for the interaction. Second, oligomerization was shown to be a prerequisite for the granular pattern of transiently expressed N protein in transfected cells. N protein trimerization was supported by three-dimensional reconstruction of the N protein by electron microscopy after negative staining. Finally, we discuss how N protein trimerization could occur.

Amino Acid Sequence↗
PubMed2002

The oligomeric state, complex formation, and chaperoning activity of hsp70 and hsp80 of Neurospora crassa.

Molecular chaperones perform vital cellular functions under normal growth conditions and protect cells against stress-induced damage. The stress proteins Hsp70 and Hsp80 of Neurospora crassa were extracted from heat-shocked mycelium, purified to near homogeneity, and examined with respect to their oligomeric state, complex formation, and chaperoning properties. Their oligomeric state was assessed by dynamic light-scattering measurements, and both Hsp70 and Hsp80 were observed to form a range of soluble, high-molecular-mass protein aggregates. Direct interaction between Hsp70 and Hsp80 was studied by partial tryptic digestion and surface plasmon resonance (SPR). Hsp70 was immobilized on the sensor chip surface, and the binding of Hsp80 in solution was followed in real time. Proteolytic digestion revealed that Hsp70-Hsp80 complex formation results in conformational changes in both proteins. The data from SPR studies yielded an equilibrium dissociation constant, KD, of 8.5 x 10(-9) M. The chaperoning ability of Hsp70, Hsp80, and Hsp70-Hsp80 was monitored in vitro by the protection of citrate synthase from thermal aggregation. The binding of nucleotides modulates the oligomeric state, chaperoning function, and hetero-oligomeric complex formation of Hsp70 and Hsp80.

Citrate (si)-Synthase↗
PubMed2004

G protein-coupled receptor oligomerization: implications for G protein activation and cell signaling.

The cardiovascular system is richly endowed with G protein-coupled receptors (GPCRs), members of the largest family of plasma membrane-localized receptors. During the last 10 years, it has become increasingly clear that many, if not all, GPCRs function in oligomeric complexes, as either homo- or hetero-oligomers. This review explores the mechanistic implications of GPCR dimerization and/or oligomerization on receptor activation and interactions with G proteins. The effects of GPCR oligomerization on receptor pharmacology, GPCR-mediated signaling, and potential contributions to GPCR crosstalk will be considered in the context of receptors important in the cardiovascular system. Our evolving understanding of the structural and functional consequences of GPCR oligomerization may provide novel and more selective sites for pharmacological tuning of cardiovascular function.

Animals↗
PubMed1998

Oligomerization of the extracellular domain of Boss enhances its binding to the Sevenless receptor and its antagonistic effect on R7 induction.

In the developing compound eye of Drosophila, neuronal differentiation of the R7 photoreceptor cell is induced by the interaction of the receptor tyrosine kinase Sevenless with its ligand Bride of sevenless (Boss), which is expressed on the neighboring R8 cell. Boss is an unusual ligand of a receptor tyrosine kinase: it is composed of a large extracellular domain, a transmembrane domain with seven membrane-spanning segments and a cytoplasmic tail. Expression of a monomeric, secreted form of the extracellular domain of Boss is not sufficient for Sevenless activation, and instead acts as a weak antagonist. Because oligomerization appears to be a critical step in the activation of receptor tyrosine kinases, we used oligomerized forms of the Boss extracellular domain to test their ability to bind to Sevenless in vivo and restore R7 induction in vivo. Oligomerization was achieved by fusion to the leucine zipper of the yeast transcription factor GCN4 or to the tetramerization helix of Lac repressor. Binding of these multivalent proteins to Sevenless could be detected in vitro by immunoprecipitation of cross-linked ligand/receptor complexes and in vivo by receptor-dependent ligand localization. However, neither R8-specific or ubiquitous expression of multivalent Exboss ligands rescued the boss phenotype. Instead, these ligands acted as competitive inhibitors for wild-type Boss protein and thereby suppressed R7 induction. Therefore the role of the transmembrane or cytoplasmic domains of Boss in the activation of the Sev receptor cannot be replaced by oligomerization.

Amino Acid Sequence↗
PubMed1998

The membrane proximal region of the integrin beta cytoplasmic domain can mediate oligomerization.

Integrin-ligand binding generates many intracellular signals, including signals to initiate focal contact formation and to regulate cellular decisions concerning growth and differentiation. Oligomerization of the beta subunit cytoplasmic domain appears to be required for many of these events. In order to study these processes, we have generated a novel chimeric protein, consisting of the chicken integrin beta 1 cytoplasmic domain connected to the central rod domain of a neuronal intermediate filament, alpha-internexin. This chimeric protein, when expressed transiently in 293T cells, oligomerizes in a beta cytoplasmic domain-dependent manner. This oligomerization requires the membrane proximal amino acids LLMII of the beta 1 cytoplasmic domain, as demonstrated by deletion analysis. Therefore, the integrin beta cytoplasmic domain in this system contains an oligomerization function, which may provide some insight as to the function of intact integrins in vivo.

Amino Acid Sequence↗
PubMed2006

Effects of signal peptide and adenylate on the oligomerization and membrane binding of soluble SecA.

SecA protein, a cytoplasmic ATPase, plays a central role in the secretion of signal peptide-containing proteins. Here, we examined effects of signal peptide and ATP on the oligomerization, conformational change, and membrane binding of SecA. The wild-type (WT) signal peptide from the ribose-binding protein inhibited ATP binding to soluble SecA and stimulated release of ATP already bound to the protein. The signal peptide enhanced the oligomerization of soluble SecA, while ATP induced dissociation of SecA oligomer. Analysis of SecA unfolding with urea or heat revealed that the WT signal peptide induces an open conformation of soluble SecA, while ATP increased the compactness of SecA. We further obtained evidences that the signal peptide-induced oligomerization and the formation of open structure enhance the membrane binding of SecA, whereas ATP inhibits the interaction of soluble SecA with membranes. On the other hand, the complex of membrane-bound SecA and signal peptide was shown to resume nucleotide-binding activity. From these results, we propose that the translocation components affect the degree of oligomerization of soluble SecA, thereby modulating the membrane binding of SecA in early translocation pathway. A possible sequential interaction of SecA with signal peptide, ATP, and cytoplasmic membrane is discussed.

Adenosine Triphosphatases↗
PubMed1999

Evidence for ligand-independent homo-oligomerization of leptin receptor (OB-R) isoforms: a proposed mechanism permitting productive long-form signaling in the presence of excess short-form expression.

The adipocyte secreted hormone leptin (OB) and its receptor (OB-R) are key regulators of mammalian body weight homeostasis. Two predominant isoforms of OB-R have been described: long form (OB-R(L)) characterized as a signal transducing receptor that is highly expressed in specific nuclei of the hypothalamus; and a short, signaling-defective form (OB-R(S)) of indeterminate function that is ubiquitously expressed throughout the body. Receptor chimera studies indicate that OB-R(L) signals via homo-oligomers. However, co-expression experiments have demonstrated that signaling by OB-R(L) is only marginally susceptible to dominant negative suppression by OB-R(S). In the present study we have used receptor epitope tagging to analyze the ligand-independent and -dependent association properties of OB-R(S) and OB-R(L). We present evidence for ligand-independent homo-oligomerization by both receptor isoforms. Ligand treatment of these complexes does not dramatically augment homo-oligomerization. In contrast, hetero-oligomerization between long and short OB-R cannot be detected in the absence of ligand but can be resolved in the presence of ligand. Deletion and substitution mutagenesis of the OB-R(L) intracellular domain indicates that ligand-independent homo-oligomerization by OB-R(L) is sensitive to reduction in JAK kinase recruitment capability, suggesting that JAK interaction and signaling competency may provide means for isoform specific OB-R sorting. These results are discussed with regard to possible mechanisms permitting efficient leptin-induced signaling by OB-R(L) in tissues that co-express OB-R(S).

Amino Acid Sequence↗
PubMed2003

Regulation of the Chk2 protein kinase by oligomerization-mediated cis- and trans-phosphorylation.

Chk2 is a serine/threonine protein kinase found mutated in certain hereditary and sporadic cancers. Ionizing radiation (IR) activates the kinase activity of Chk2 in a phosphorylation-dependent manner. ATM phosphorylates Chk2 on threonine 68, which promotes oligomerization and phosphorylation on threonines 383 and 387 within the activation loop of the catalytic domain. In this study, threonines 68, 383, and 387 were confirmed as sites of Chk2 phosphorylation both in vitro and in vivo. In addition, serine 516 was identified as a novel IR-inducible phosphorylation site in vivo and as a site of autophosphorylation in vitro. Interestingly, Chk2 was capable of autoactivation in the absence of IR when overproduced in bacteria, in 293 cells, and in murine embryonic fibroblasts lacking Chk2. A kinase-inactive mutant of Chk2 was phosphorylated on T68 and T383/T387 but not on S516 in cells containing Chk2 and on T68 but not T383/T387 or S516 in cells lacking Chk2. This establishes a dependency on Chk2 kinase activity for phosphorylation of T383/T387 and S516 but not for T68 in vivo. We demonstrate that T68 phosphorylation is regulated by kinases in addition to ATM and Chk2. Taken together, our data indicate that autophosphorylation of Chk2 can occur both in cis and in trans and suggest that oligomerization may regulate Chk2 activation by promoting these cis- and trans-phosphorylation events. The importance of oligomerization is underscored by the observation that substitution of isoleucine for threonine at position 157, a mutation found in a subset of patients with Li-Fraumeni syndrome, impairs both Chk2 oligomerization and autophosphorylation.

Bacteria↗
PubMedDate not supplied

Cryo-bioorganic chemistry: freezing effect on stereoselection of DL-alanine-N-carboxyanhydride oligomerization in dioxane solution.

The possibility of stereoselection through the DL-alanine-N-carboxyanhydride (NCA-DL-Ala) oligomerization, and the effect of freezing on it have been investigated. To this end, the chirality of peptides obtained by oligomerization for 1 and 3 days, respectively, in liquid (+22 degree celsius) and frozen (-18 degree celsius) dioxane solutions, was analyzed. These water-soluble samples were fractionated by gel filtration, aliquots of the fractions were completely hydrolyzed and then derivatized with Marfey reagent (1-fluoro-2,4-dinitrophenyl-5-L-alanine amide). These derivatives were traced and evaluated by RP-HPLC analysis. The relatively best effects appeared in a given fraction, where after 1 day of oligomerization the L-alanine enantiomeric excess (ee) was 3.8 percent in liquid and 8.6 percent in frozen conditions. After 3 days, however, the ee contents decreased to 2.0 percent and 4.1 percent. The mass spectrometric data of the peptides pointed to the formation of open chain and cyclic peptide mixtures, where the residue numbers of 8-11 and 4-5 dominated. The formation of some chiral peptides from a racemic amino acid suggests the possibility of preferential incorporation of the L-enantiomer into the growing chain, beside the achiral statistical succession of residues. Here we provide the first example of the role of freezing in the increased formation of chirality from racemic amino-acid through oligomerization, together with some speculations about the implications of our model in the events of prebiotic chemistry.

Alanine↗
PubMed2005

Antiviral activity of oligomerization-deficient Stat1.

Interferon stimulation of cells can activate several hundred target genes, many of which are required for antiviral protection. Promoter binding of tyrosine-phosphorylated (activated) Stat1 dimers is essential for gene induction, a process that often entails the oligomerization of Stat1 dimers via interactions of their aminoterminal domains. The mutation of a single residue (F77) in the N-domain of Stat1 was recently demonstrated to preclude both the dephosphorylation and the oligomerization of Stat1 dimers. Here, we investigated the influence of defective oligomerization on a complex phenotype such as the induction of an antiviral state. It was found that the antiviral protection conferred by interferon-a was strongly reduced, whereas the interferon-g response was not measurably affected. These results indicate that Stat1 oligomerization is required for the antiviral activity of interferons. Moreover, the concentration of activated Stat1 in the nucleus may generally play a critical role for interferon-induced target gene activation.

Antiviral Agents↗