RESUMO
Actin-based gels were prepared from clarified high-salt extracts of human platelets by dialysis against physiological salt buffers. The gel was partially solubilized with 0.3 M KCl. Mice were immunized with the 0.3 M KCl extract of the actin gel, and hybridomas were produced by fusion of spleen cells with myeloma cells. Three hybridomas were generated that secrete antibodies against an 80-kD protein. These monoclonal antibodies stained stress fibers in cultured cells and cross-reacted with proteins in several tissue types, including smooth muscle. The cross-reacting protein in chicken gizzard smooth muscle had an apparent molecular weight of 140,000 and was demonstrated to be caldesmon, a calmodulin and actin-binding protein (Sobue, K., Y. Muramoto, M. Fujita, and S. Kakiuchi, Proc. Natl. Acad. Sci. USA, 78:5652-5655). No proteins of molecular weight greater than 80 kD were detectable in platelets by immunoblotting using the monoclonal antibodies. The 80-kD protein is heat stable and was purified using modifications of the procedure reported by Bretscher for the rapid purification of smooth muscle caldesmon (Bretscher, A., 1985, J. Biol. Chem., 259:12873-12880). The 80-kD protein bound to calmodulin-Sepharose in a Ca++-dependent manner and sedimented with actin filaments, but did not greatly increase the viscosity of F-actin solutions. The actin-binding activity was inhibited by calmodulin in the presence of calcium. Except for the molecular weight difference, the 80-kD platelet protein appears functionally similar to 140-kD smooth muscle caldesmon. We propose that the 80-kD protein is platelet caldesmon.
Assuntos
Anticorpos Monoclonais/imunologia , Plaquetas/análise , Proteínas de Ligação a Calmodulina/imunologia , Proteínas de Transporte/imunologia , Proteínas dos Microfilamentos/imunologia , Animais , Calmodulina/metabolismo , Galinhas , Imunofluorescência , Géis , Gelsolina , Moela das Aves/análise , Humanos , Peso MolecularRESUMO
A 130,000 Mr protein was isolated from human platelets by sequential DEAE-Sephacel and Sepharose Cl-4B chromatography. Low shear viscometric measurements showed that the enriched protein after DEAE-Sephacel chromatography inhibited actin polymerization. This effect was somewhat greater in the presence of EGTA than in the presence of calcium. Further purification by Sepharose Cl-4B chromatography resulted in a complete loss of this inhibitory effect. Studies with fluorescent actin detected no nucleation or "+" end capping activity in either the DEAE-Sephacel- or Sepharose Cl-4B-purified vinculin. Antibodies raised in mice against the 130,000-mol-wt protein were shown to cross-react with chicken gizzard vinculin and a similar molecular weight protein was detected in WI38 cells and, Madin-Darby canine kidney cells. Lysis experiments with the Madin-Darby canine kidney cells indicated that most of the vinculin was soluble in Triton X-100, although some was found associated with the insoluble cytoskeletal residue. By immunofluorescence, vinculin in WI38 cells was localized to adhesion plaques as described by others. Discrete localization in platelets was also detected and appeared to depend on their state of adhesion and spreading. The results of these experiments suggest that human platelets contain a protein similar to vinculin. It is not clear if platelet vinculin is associated with structures analogous to adhesion plaques found in other cell types. The data indicate that the previously reported effects of nonmuscle vinculins on actin polymerization may be due to a contaminant or contaminants.
Assuntos
Plaquetas/análise , Proteínas Musculares/sangue , Actinas , Especificidade de Anticorpos , Células Cultivadas , Reações Cruzadas , Citoesqueleto/análise , Humanos , Peso Molecular , Proteínas Musculares/imunologia , Vinculina , ViscosidadeRESUMO
Gelsolin is a 90,000-mol-wt Ca2+-binding, actin-associated protein that can nucleate actin filament growth, sever filaments, and cap barbed filament ends. Brevin is a closely related 92,000-mol-wt plasma protein with similar properties. Gelsolin has been reported to be localized on actin filaments in stress fibers, in cardiac and skeletal muscle I-bands, and in cellular regions where actin filaments are known to be concentrated. Previous localization studies have used sera or antibody preparations that contain brevin. Using purified brevin-free IgG and IgA monoclonal antibodies or affinity-purified polyclonal antibodies for gelsolin and brevin, we find no preferential stress fiber staining in cultured human fibroblasts or I-band staining in isolated rabbit skeletal muscle sarcomeres. Cardiac muscle frozen sections show no pronounced I-band staining, except in local areas where brevin may have penetrated from adjacent blood vessels. Spreading platelets show endogenous gelsolin localized at the cell periphery, in the central cytoplasmic mass and on thin fibers that radiate from the central cytoplasm. Addition of 3-30 micrograms/ml of brevin to the antibodies restores intense stress fiber and I-band staining. We see no evidence for large-scale severing and removal of filaments in stress fibers in formaldehyde-fixed, acetone-permeabilized cells even at brevin concentrations of 30 micrograms/ml. The added brevin or brevin antibody complex binds to actin filaments and is detected by the fluorescently tagged secondary antibody. Brevin binding occurs in either Ca2+ or EGTA, but is slightly more intense in EGTA suggesting some severing and filament removal may occur in Ca2+. The I-band staining is limited to the region where actin and myosin do not overlap. In addition, brevin does not appear to bind at the Z-line. A comparison of cells double-labeled with fluorescein-phallotoxin, exogenous brevin, and a monoclonal antibody, detected with a rhodamine-labeled secondary antibody, shows almost complete co-localization of F-actin with the brevin-gelsolin-binding sites. A major exception is in the area of the adhesion plaque. A quantitative comparison of the fluorescein-rhodamine fluorescence intensities along a stress fiber and into the adhesion plaque shows that the fluorescein signal, associated with F-actin, increases while the rhodamine signal decreases. We infer that exogenous brevin or endogenous gelsolin can bind to and potentially sever most actin filaments, but that actin-associated proteins in the adhesion plaque can prevent binding and severing.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Citoesqueleto/ultraestrutura , Proteínas dos Microfilamentos/metabolismo , Actinas/metabolismo , Sítios de Ligação , Plaquetas/ultraestrutura , Proteínas de Transporte/metabolismo , Células Cultivadas , Imunofluorescência , Gelsolina , Humanos , Proteínas Musculares/metabolismo , Músculos/ultraestrutura , Miocárdio/metabolismo , Miocárdio/ultraestruturaRESUMO
Receptor-mediated activation of heterotrimeric guanine nucleotide-binding proteins (G proteins) results in the dissociation of alpha from beta gamma subunits, thereby allowing both to regulate effectors. Little is known about the regions of effectors required for recognition of G beta gamma. A peptide encoding residues 956 to 982 of adenylyl cyclase 2 specifically blocked G beta gamma stimulation of adenylyl cyclase 2, phospholipase C-beta 3, potassium channels, and beta-adrenergic receptor kinase as well as inhibition of calmodulin-stimulated adenylyl cyclases, but had no effect on interactions between G beta gamma and G alpha o. Substitutions in this peptide identified a functionally important motif, Gln-X-X-Glu-Arg, that is also conserved in regions of potassium channels and beta-adrenergic receptor kinases that participate in G beta gamma interactions. Thus, the region defined by residues 956 to 982 of adenylyl cyclase 2 may contain determinants important for receiving signals from G beta gamma.
Assuntos
Adenilil Ciclases/química , Proteínas de Ligação ao GTP/fisiologia , Inibidores de Adenilil Ciclases , Adenilil Ciclases/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Ativação Enzimática/fisiologia , Proteínas de Ligação ao GTP/química , Guanosina Trifosfato/fisiologia , Técnicas In Vitro , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/fisiologia , Canais de Potássio/fisiologia , Ratos , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Adrenérgicos beta/metabolismo , Transdução de Sinais/fisiologia , Relação Estrutura-Atividade , Fosfolipases Tipo C/metabolismoRESUMO
Three products of the MAP2 gene are known: MAP2A and MAP2B (Mr approximately 200,000) and MAP2C (Mr 70,000). The structural relationship between these MAPs and the basis for their diversity in size are unknown. Previously, we found that a significant fraction of type II cAMP-dependent protein kinase was associated via its regulatory subunits with MAP2A and MAP2B. We now use an antibody prepared against the microtubule binding domain of MAP2A and MAP2B to identify MAP2C. All three forms of MAP2 bound to cAMP affinity columns and reacted with 32P-labeled RII in a blot overlay assay. By assaying proteolytic fragments of MAP2A and MAP2B as well as segments of MAP2 expressed in E. coli, the binding site for RII was localized to an 83 amino acid stretch at the distal (amino-terminal) end of the MAP2 arm domain. Therefore, the microtubule binding and RII binding domains are located at extreme opposite ends of MAP2A and MAP2B, and both are conserved in the much shorter MAP2C.
Assuntos
Proteínas de Transporte/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Quinases/metabolismo , Animais , Anticorpos/imunologia , Sítios de Ligação , Fenômenos Químicos , Química , Cromatografia de Afinidade , Reações Cruzadas , AMP Cíclico/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/imunologiaRESUMO
The heterotrimeric GTP binding proteins, G proteins, consist of three distinct subunits: alpha, beta, and gamma. There are 12 known mammalian gamma subunit genes whose products are the smallest and most variable of the G protein subunits. Sequencing of the bovine brain gamma(10) protein by electrospray mass spectrometry revealed that it differs from the human protein by an Ala to Val substitution near the N-terminus. Comparison of gamma isoform subunit sequences indicated that they vary substantially more at the N-terminus than at other parts of the protein. Thus, species variation of this region might reflect the lack of conservation of a functionally unimportant part of the protein. Analysis of 38 gamma subunit sequences from four different species shows that the N-terminus of a given gamma subunit isoform is as conserved between different species as any other part of the protein, including highly conserved regions. These data suggest that the N-terminus of gamma is a functionally important part of the protein exhibiting substantial isoform-specific variation.
Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/química , Alanina/química , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Sequência Conservada , Etiquetas de Sequências Expressas , Proteínas Heterotriméricas de Ligação ao GTP/genética , Humanos , Filogenia , Ligação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Valina/químicaAssuntos
Encéfalo/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Animais , Proteínas de Bactérias , Biotina , Bovinos , Cromatografia de Afinidade/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/isolamento & purificação , Indicadores e Reagentes , Substâncias Macromoleculares , Peso Molecular , Ligação Proteica , EstreptavidinaRESUMO
Members of the heat-stable family of microtubule-associated proteins (MAPs), MAP 2, tau, and MAP 4, contain three or four tandem imperfect repeated sequences close to their carboxyl termini. These sequences lie within the microtubule-binding domains of the MAPs; they have been proposed to be responsible for microtubule binding and the ability of these MAPs to lower the critical concentration for microtubule assembly. Their spacing may reflect that of the regularly arrayed tubulin subunits on the microtubule surface. We here characterize the 32- and 34-kDa chymotryptic microtubule-binding fragments of MAP 2 identified in earlier work. We identify the primary chymotryptic cleavage site in high molecular weight MAP 2 as between Phe1525 and Lys1526, within 13 amino acids of the known MAP 2 splice junction. We have raised a monoclonal antibody to the 32- and 34-kDa fragments and find that it reacts with all members of the heat-stable MAPs class. To determine where it reacts, we sequenced immunoreactive subfragments of the 32- and 34-kDa fragments, selected several cDNA clones with the antibody, and tested for antibody reactivity against a series of synthetic MAP 2 and tau peptides. We identify the epitope sequence as HHVPGGG (His-His-Val-Pro-Gly-Gly-Gly). The antibody also recognized several other MAP 2 and tau repeats. Despite reacting with this highly conserved element, we find that the antibody does not block microtubule binding, but binds to the MAPs and co-sediments with microtubules. These results suggest that there are other regions besides the repeated elements which are essential for microtubule binding.
Assuntos
Anticorpos Monoclonais/biossíntese , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Reações Cruzadas , Epitopos , Temperatura Alta , Immunoblotting , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/imunologia , Dados de Sequência MolecularRESUMO
The region encoded by amino acids 956-982 of adenylyl cyclase 2 is important for Gbetagamma stimulation. Interactions of a peptide encoding the 956-982 region of adenylyl cyclase 2 (QEHAQEPERQYMHIGTMVEFAYALVGK (QEHA peptide)) with Gbetagamma subunits were studied. QEHA peptide was covalently attached to beta subunit of free Gbetagamma by the cross-linker N-succinimidyl(4-iodoacetyl)aminobenzoate. Cross-linking was proportional to the amount of QEHA peptide added; other control peptides cross-linked minimally. When Go was used, very little cross-linking was observed with GDP and EDTA, but upon activation by guanosine 5'-3-O-(thio)triphosphate and Mg2+, specific cross-linking of the QEHA peptide to Gbeta was observed. We conclude that beta subunits of G proteins contain effector interaction domains that are occluded by Galpha subunits in the heterotrimer. Molecular modeling studies used to dock the QEHA peptide on to Gbeta indicate that amino acids 75-165 of Gbeta may be involved in effector interactions.
Assuntos
Adenilil Ciclases/química , Proteínas de Ligação ao GTP/metabolismo , Fragmentos de Peptídeos/metabolismo , Sequência de Aminoácidos , Reagentes de Ligações Cruzadas , Proteínas de Ligação ao GTP/química , Dados de Sequência Molecular , SuccinimidasRESUMO
Among subfamilies of G-protein-coupled receptors, agonists initiate several cell signaling events depending on the receptor subtype (R) and the type of G-protein (G) or effector molecule (E) expressed in a particular cell. Determinants of signaling specificity/efficiency may operate at the R-G interface, where events are influenced by cell architecture or accessory proteins found in the receptor's microenvironment. This issue was addressed by characterizing signal transfer from R to G following stable expression of the alpha 2A/D adrenergic receptor in two different membrane environments (NIH-3T3 fibroblasts and the pheochromocytoma cell line, PC-12). Receptor coupling to endogenous G-proteins in both cell types was eliminated by pertussis toxin pretreatment and R-G signal transfer restored by reconstitution of cell membranes with purified brain G-protein. Thus, the receptor has access to the same population of G-proteins in the two different environments. In this signal restoration assay, agonist-induced activation of G was 3-9-fold greater in PC-12 as compared with NIH-3T3 alpha 2-adrenergic receptor transfectants. The cell-specific differences in signal transfer were observed over a range of receptor densities or G-protein concentration. The augmented signal transfer in PC-12 versus NIH-3T3 transfectants occurred despite a 2-3-fold lower level of receptors existing in the R-G-coupled state (high affinity, guanyl-5'-yl imidodiphosphate-sensitive agonist binding), suggesting the existence of other membrane factors that influence the nucleotide binding behavior of G-protein in the two cell types. Detergent extraction of PC-12 but not NIH-3T3 membranes yielded a heat-sensitive, macromolecular entity that increased 35S-labeled guanosine 5'-O-(thiotriphosphate) binding to brain G-protein in a concentration-dependent manner. These data indicate that the transfer of signal from R to G is regulated by a cell type-specific, membrane-associated protein that enhances the agonist-induced activation of G.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Transdução de Sinais , Células 3T3 , Agonistas alfa-Adrenérgicos/metabolismo , Animais , Encéfalo/metabolismo , Tartarato de Brimonidina , Bovinos , Linhagem Celular , Membrana Celular/metabolismo , Proteínas de Ligação ao GTP/biossíntese , Proteínas de Ligação ao GTP/isolamento & purificação , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanilil Imidodifosfato/farmacologia , Cinética , Camundongos , Células PC12 , Toxina Pertussis , Quinoxalinas/metabolismo , Ratos , Receptores Adrenérgicos alfa 2/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Transfecção , Fatores de Virulência de Bordetella/farmacologiaRESUMO
The G protein gamma5 subunit is selectively associated with specific G protein alpha subunits [Wilcox, M. D., et al. (1995) J. Biol. Chem. 270, 4189] and is localized preferentially in focal adhesion plaques [Hansen, C. A., et al. (1996) J. Cell Biol. 126, 811]. What determines the differential association of G proteins and their subunits with specific cellular structures or compartments is not clear, but one factor could be variation in the pattern of processing of the proteins. To study gamma5 subunit diversity and modifications, G protein subunits were fractionated on an HPLC phenyl column and analyzed with a gamma5-specific antiserum. The gamma5 eluted from the column as two peaks of immunoreactivity. Analysis by matrix-assisted laser desorption ionization (MALDI) mass spectrometry and electrospray ionization tandem mass spectrometry revealed that the first immunoreactive peak corresponded to the predicted gamma5 isoform (N-terminally acetylated after removal of methionine, C-terminally geranylgeranylated and carboxymethylated with removal of the last three amino acids), while the second peak of immunoreactivity contained a gamma5 isoform isoprenylated at the C-terminus but retaining its three terminal amino acids. This alternatively processed protein is the predominant gamma5 subunit isoform associated with Go and Gi proteins purified from bovine brain. These results describe a new C-terminal processing pattern for G protein gamma subunits and establish the principle that G protein gamma subunits can be heterogeneously modified at their C-termini. This is a site on the gamma subunit critical for membrane and protein-protein interactions of G proteins. These results open the possibility that one determinant of the localization of G proteins in cells could be the pattern of processing of their gamma subunit constituents.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Ácido Aspártico/metabolismo , Bovinos , Membrana Celular/química , Membrana Celular/metabolismo , Córtex Cerebral/química , Córtex Cerebral/metabolismo , Cromatografia Líquida de Alta Pressão , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/isolamento & purificação , Hidrólise , Immunoblotting , Isomerismo , Espectrometria de Massas , Prolina/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Extracts of human platelets contain a 90,000-Da protein that is retained by DNase I-agarose in the presence of Ca2+. The 90-kDa protein, tightly complexed with platelet actin, can be eluted from DNase I-agarose by ethylene glycol bis(beta-aminoethyl ether)-N, N,N',N'-tetraacetic acid (EGTA). The platelet 90-kDa protein is immunologically related to rabbit macrophage gelsolin. The 90-kDa protein-actin complex was purified from platelet extracts using DEAE-Sephacel, Sephadex G-200, and hydroxyapatite and is stable in EGTA and 0.8 M KCl. The purified complex will modulate the assembly of fluorescently labeled 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole-actin in the presence of both Ca2+ and EGTA. In addition, the complex affects the low shear viscosity of F-actin solutions in the presence of both Ca2+ and EGTA. Finally, the complex increases the critical concentration for actin assembly about 4-fold. The results are consistent with a strong preferential binding to or capping of the barbed end of actin filaments by the complex in either Ca2+ or EGTA.
Assuntos
Actinas/isolamento & purificação , Plaquetas/análise , Proteínas de Ligação ao Cálcio/isolamento & purificação , Cálcio/sangue , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Ácido Egtázico , Gelsolina , Humanos , Substâncias Macromoleculares , Peso MolecularRESUMO
The distribution and properties in brain of the alpha subunits of the major bovine brain Go isoforms, GoA, GoB and GoC, were characterized. The alpha(o)A and alpha(o)B isoforms arise from alternative splicing of RNAs from a single alpha(o) gene, whereas alpha(o)C is a deamidated form of alpha(o)A. All three Go isoforms purify from brain with different populations of betagamma dimers. This variable subunit composition of Go heterotrimers is likely a consequence of their functional differences. This study examined the biochemical properties of the alpha(o) isoforms to see if these properties explain the variable betagamma composition of their heterotrimers. The brain distribution of alpha(o)B differed substantially from that of alpha(o)A and alpha(o)C, as did its guanine nucleotide binding properties. The unique subunit composition of GoB can be explained by its expression in different brain regions. The alpha(o)A and alpha(o)C showed slight differences in guanine nucleotide binding properties but no preference for particular betagamma dimers when reassociated with a heterogeneous betagamma pool. The alpha(o)C protein occurred in a constant ratio to alpha(o)A throughout the brain, but was a much larger percent of total brain alpha(o) than previously thought, approximately 35%. These results suggest that alpha(o)A is a precursor of alpha(o)C and that the association of G(o)alpha subunits with different betagamma dimers reflects the function of an adaptive, G-protein signaling mechanism in brain.
Assuntos
Química Encefálica , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Animais , Anticorpos , Ligação Competitiva/fisiologia , Bovinos , Quelantes/farmacologia , Dimerização , Ácido Edético/farmacologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP , Proteínas de Ligação ao GTP/imunologia , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Guanosina Difosfato/farmacologia , Immunoblotting , Isomerismo , Cloreto de Magnésio/farmacologia , Memória/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Go is the major G protein in bovine brain, with at least three isoforms, GoA, GoB, and GoC. Whereas alphaoA and alphaoB arise from a single Goalpha gene as alternatively spliced mRNAs, alphaoA and alphaoC are thought to differ by covalent modification. To test the hypothesis that alphaoA and alphaoC have different N-terminal lipid modifications, proteolytic fragments of alphao isoforms were immunoprecipitated with an N terminus-specific antibody and analyzed by matrix-assisted laser desorption ionization mass spectrometry. The major masses observed in immunoprecipitates were the same for all three alphao isoforms and corresponded to the predicted mass of a myristoylated N-terminal fragment. Structural differences between alphaoA and alphaoC were also compared before and after limited tryptic proteolysis using SDS-polyacrylamide gel electrophoresis containing 6 M urea. Based upon the alphao subunit fragments produced under activating and nonactivating conditions, differences between alphaoA and alphaoC were localized to a C-terminal fragment of the protein. This region, involved in receptor and effector interactions, implies divergent signaling roles for these two alphao proteins. Finally, the structural difference between alphaoA and alphaoC is associated with a difference of at most 2 daltons based upon measurements by electrospay ionization mass spectrometry.
Assuntos
Encéfalo/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Proteínas de Ligação ao GTP/química , Isomerismo , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Receptores de Superfície Celular/metabolismoRESUMO
We have studied the microtubule-associated protein MAP2 from porcine brain and its subfragments by limited proteolysis, antibody labeling, and electron microscopy. Two major chymotryptic fragments start at lys 1528 and arg 1664, generating microtubule-binding fragments of Mr 36 kDa (303 residues, analogous to the "assembly domain" of Vallee, 1980) and 18 kDa (167 residues). These fragments can be labeled with the antibody 2-4 which recognizes the last internal repeat of MAP2 (Dingus et al., 1991). The epitope of another monoclonal antibody, AP18 (Binder et al., 1986), was mapped to the first 151 residues of MAP2. The interaction with AP18 is phosphorylation dependent; dephosphorylated MAP2 is not recognized. Intact MAP2 forms rod-like particles of 97 nm mean length, similar to Gottlieb and Murphy's (1985) observations. Both antibodies bind near an end of the rod, suggesting that the sequence and the structure are approximately colinear. There is a pronounced tendency for MAP2 to form dimers whose components are nearly in register but of opposite polarity. MAP2 can also fold in a hairpin-like fashion, generating 50-nm rods, and it can self-associate into oligomers and fibers. The 36-kDa microtubule-binding fragment also has a rod-like shape; its mean length is 49 nm, half of the intact molecule, even though the fragment contains only one-sixth of the mass. The antibody 2-4 decorates one end of the rod, similar to the intact protein. The fragment also forms antiparallel dimers, but its tendency for higher self-assembly forms is much lower than with intact MAP2.
Assuntos
Proteínas Associadas aos Microtúbulos/ultraestrutura , Sequência de Aminoácidos , Animais , Bovinos , Eletroforese em Gel de Poliacrilamida , Epitopos , Magnésio/farmacologia , Microscopia Eletrônica , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/imunologia , Dados de Sequência Molecular , Fosforilação , SuínosRESUMO
Receptor activation of heterotrimeric G proteins dissociates G alpha from the G betagamma complex, allowing both to regulate effectors. Little is known about the effector-interaction regions of G betagamma. We had used molecular modeling to dock a peptide encoding the region of residues 956-982 of adenylyl cyclase (AC) 2 onto Gbeta to identify residues on Gbeta that may interact with effectors. Based on predictions from the model, we synthesized peptides encoding sequences of residues 86-105 (Gbeta 86-105) and 115-135 (Gbeta 115-135) from Gbeta. The Gbeta 86-105 peptide inhibited G betagamma stimulation of AC2 and blocked G betagamma inhibition of AC1 and by itself inhibited calmodulin-stimulated AC1, thus displaying partial agonist activity. Substitution of Met-101 with Asn in this peptide resulted in the loss of both the inhibitory and partial agonist activities. Most activities of the Gbeta 115-135 peptide were similar to those of Gbeta 86-105 but Gbeta 115-135 was less efficacious in blocking G betagamma inhibition of AC1. Substitution of Tyr-124 with Val in the Gbeta 115-135 peptide diminished all of its activities. These results identify the region encoded by amino acids 84-143 of Gbeta as a surface that is involved in transmitting signals to effectors.
Assuntos
Adenilil Ciclases/química , Adenilil Ciclases/metabolismo , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Conformação Proteica , Adenilil Ciclases/biossíntese , Sequência de Aminoácidos , Animais , Sítios de Ligação , Encéfalo/metabolismo , Bovinos , Proteínas de Ligação ao GTP/biossíntese , Substâncias Macromoleculares , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Biossíntese de Proteínas , Estrutura Secundária de Proteína , Coelhos , Reticulócitos/metabolismoRESUMO
The diversity of the gamma subunits in bovine brain G protein preparations was investigated using matrix-assisted laser desorption ionization (MALDI) mass spectrometry. Analysis of these G protein mixtures revealed at least four gamma subunit masses by the following four criteria. 1) The measured masses were in the same mass range as the predicted molecular weights of gamma isoforms. 2) The masses were reproducible between the same or different preparations of G proteins. 3) The masses were independent of the matrix used for MALDI analysis. 4) The masses comigrated with the gamma subunit, as part of the heterotrimer, the beta gamma dimer, or the separated gamma subunit. These measured masses were compared with those calculated from cDNA sequences of known bovine brain gamma isoforms with the addition of plausible post-translational modifications. The mass of each spectral peak was consistent with the calculated mass for only one of four known bovine brain gamma subunit isoforms, but the data suggest modifications of the gamma subunits in addition to those already known or suspected at their carboxyl termini. Besides these four major masses, several additional, less resolved spectral peaks were observed whose measured masses did not correlate with any known gamma subunit or plausible modification. MALDI mass spectrometry promises to be a powerful technique for the analysis of the diversity of the gamma subunit in G proteins and for the characterization of their post-translational modifications.
Assuntos
Proteínas de Ligação ao GTP/química , Animais , Química Encefálica , Bovinos , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Proteínas de Ligação ao GTP/isolamento & purificação , Espectrometria de Massas , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificaçãoRESUMO
The gamma subunit composition of the major bovine brain Go and Gi proteins (GOA, GOB, GOC, Gi1, and Gi2) was characterized using antibodies against specific gamma isoforms. Each of the purified G protein heterotrimers contained a heterogeneous population of gamma subunits, and the profiles of the gamma subunits found with Gi1, Gi2, and GOA were similar. In contrast, each GO isoform had a distinct pattern of associated gamma subunits. These differences were surprising given that all three alpha O isoforms are thought to share a common amino-terminal sequence important for the binding of beta gamma dimers and that the alpha OA and alpha OC proteins may come from the same alpha O1 mRNA. The free alpha OA and alpha OC subunits had unique elution behaviors during MonoQ chromatography, compatible with differences in their post-translational processing. These results indicate that both the alpha and gamma subunit compositions of heterotrimers define the structure of an intact G protein. Furthermore, the exact subunit composition of G protein heterotrimers may depend upon regulated expression of different subunit isoforms or upon cellular processing of alpha subunits.