Gamma-subunits of G proteins, but not their alpha- or beta-subunits, are polyisoprenylated. Studies on post-translational modifications using in vitro translation with rabbit reticulocyte lysates

Considerable advances have been made in the genetics of salivary proline-rich proteins (PRP). The genes for acidic, basic, and glycosylated PRP have been cloned. They code for precursor proteins that all have an acidic N-terminal followed by proline-rich repeat sequences. Structural studies on secreted proteins have demonstrated that not only acidic but also some basic PRPs have this general structure. It is possible that mRNA for different PRP may have originated from a single gene by differential mRNA splicing, but post-translational cleavages of the primary translation product apparently also occur. In vitro translation of salivary gland mRNA results in a single precursor protein for acidic PRP. Such in vitro translated protein can be cleaved by salivary kallikrein, giving rise to two commonly secreted acidic PRPs, and kallikrein or kallikrein-like enzymes may be responsible for other post-translational cleavages of PRPs. Acidic as well as some basic PRPs are phosphorylated. A protein kinase has been demonstrated in salivary glands which phosphorylates the PRPs and other secreted salivary proteins in a cAMP and Ca2+-calmodulinindependent manner. Knowledge of the conformation of PRPs is limited. There is no conclusive evidence of polyproline-like structure in the proline-rich part of PRPs. Ca2+ binding studies on acidic PRPs indicate that there is interaction between the Ca2+ binding N-terminal end and the proline-rich C-terminal part. This interaction is relieved by modification of arginine side-chains. 1H, 32P, and 43Ca NMR studies have further elucidated the conformation of acidic PRPs in solution. Present evidence shows that salivary PRPs constitute a unique superfamily of proteins which pose a number of interesting questions concerning gene structure, pre- and post-translational modifications, and protein conformation.

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Differential distribution of alpha subunits and beta gamma subunits of heterotrimeric G proteins on Golgi membranes of the exocrine pancreas.

The Journal of Cell Biology ◽

10.1083/jcb.133.5.1027 ◽

1996 ◽

Vol 133 (5) ◽

pp. 1027-1040 ◽

Cited By ~ 100

Author(s):

S P Denker ◽

J M McCaffery ◽

G E Palade ◽

P A Insel ◽

M G Farquhar

Keyword(s):

G Proteins ◽

Exocrine Pancreas ◽

Protein S ◽

Beta Subunits ◽

Heterotrimeric G Proteins ◽

Differential Distribution ◽

Alpha Subunits ◽

Golgi Membranes ◽

Gamma Subunits ◽

G Alpha Q

Heterotrimeric G proteins are well known to be involved in signaling via plasma membrane (PM) receptors. Recent data indicate that heterotrimeric G proteins are also present on intracellular membranes and may regulate vesicular transport along the exocytic pathway. We have used subcellular fractionation and immunocytochemical localization to investigate the distribution of G alpha and G beta gamma subunits in the rat exocrine pancreas which is highly specialized for protein secretion. We show that G alpha s, G alpha i3 and G alpha q/11 are present in Golgi fractions which are > 95% devoid of PM. Removal of residual PM by absorption on wheat germ agglutinin (WGA) did not deplete G alpha subunits. G alpha s was largely restricted to TGN-enriched fractions by immunoblotting, whereas G alpha i3 and G alpha q/11 were broadly distributed across Golgi fractions. G alpha s did not colocalize with TGN38 or caveolin, suggesting that G alpha s is associated with a distinct population of membranes. G beta subunits were barely detectable in purified Golgi fractions. By immunofluorescence and immunogold labeling, G beta subunits were detected on PM but not on Golgi membranes, whereas G alpha s and G alpha i3 were readily detected on both Golgi and PM. G alpha and G beta subunits were not found on membranes of zymogen granules. These data indicate that G alpha s, G alpha q/11, and G alpha i3 associate with Golgi membranes independent of G beta subunits and have distinctive distributions within the Golgi stack. G beta subunits are thought to lock G alpha in the GDP-bound form, prevent it from activating its effector, and assist in anchoring it to the PM. Therefore the presence of free G alpha subunits on Golgi membranes has several important functional implications: it suggests that G alpha subunits associated with Golgi membranes are in the active, GTP-bound form or are bound to some other unidentified protein(s) which can substitute for G beta gamma subunits. It further implies that G alpha subunits are tethered to Golgi membranes by posttranslational modifications (e.g., palmitoylation) or by binding to another protein(s).

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N-terminal binding domain of Gα subunits: involvement of amino acids 11–14 of Gα0 in membrane attachment

Biochemical Journal ◽

10.1042/bj3230239 ◽

1997 ◽

Vol 323 (1) ◽

pp. 239-244 ◽

Cited By ~ 5

Author(s):

Liliana BUSCONI ◽

Paula M. BOUTIN ◽

Bradley M. DENKER

Keyword(s):

Signal Transduction ◽

Amino Acids ◽

G Proteins ◽

Membrane Binding ◽

Amino Acid Sequences ◽

Membrane Receptors ◽

In Vitro Translation ◽

Membrane Attachment ◽

Membrane Components

Heterotrimeric guanine nucleotide binding proteins (G-proteins) transmit signals from membrane receptors to a variety of intracellular effectors. G-proteins reversibly associate with components of the signal transduction system, yet remain membrane attached throughout the cycle of activation. The Gα subunits remain attached to the plasma membrane through a combination of factors that are only partially defined. We now demonstrate that amino acids within the N-terminal domain of Gα subunits are involved in membrane binding. We used in vitro translation, a technique widely utilized to characterize functional aspects of G-proteins, and interactions with donor-acceptor membranes to demonstrate that amino acids 11-14 of Gαo contribute to membrane binding. The membrane binding of Gαo lacking amino acids 11-14 (D[11-14]) was significantly reduced at all membrane concentrations in comparison with wild-type Gαo. Several other N-terminal mutants of Gαo were characterized as controls, and these results indicate that differences in myristoylation, palmitoylation and βγ interactions do not account for the reduced membrane binding of D[11-14]. Furthermore, when membrane attachment of Gαo and mutants was characterized in transiently transfected 35S-labelled and [3H]myristate-labelled COS cells, amino acids 11-14 contributed to membrane binding. These studies reveal that membrane binding of Gα subunits occurs by a combination of factors that include lipids and amino acid sequences. These regions may provide novel sites for interaction with membrane components and allow additional modulation of signal transduction.

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XENOPUS FIBRINOGEN: CHARACTERIZATION OF SUBUNITS, POST-TRANSLATIONAL MODIFICATIONS, IN VITRO TRANSLATION, AND GLUCOCORTICOID-REGULATED SYNTHESIS

Annals of the New York Academy of Sciences ◽

10.1111/j.1749-6632.1983.tb23287.x ◽

1983 ◽

Vol 408 (1 Molecular Bio) ◽

pp. 649-650

Author(s):

Lené J. Holland ◽

John W. Weisel ◽

Lawrence J. Wangh

Keyword(s):

In Vitro Translation ◽

Post Translational Modifications

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Biochemical and genetic analysis of dominant-negative mutations affecting a yeast G-protein gamma subunit.

Molecular and Cellular Biology ◽

10.1128/mcb.14.7.4571 ◽

1994 ◽

Vol 14 (7) ◽

pp. 4571-4578 ◽

Cited By ~ 26

Author(s):

A V Grishin ◽

J L Weiner ◽

K J Blumer

Keyword(s):

G Protein ◽

Dominant Negative ◽

Beta Subunits ◽

Yeast Saccharomyces Cerevisiae ◽

Surface Receptors ◽

Receptor Coupling ◽

Gamma Subunits ◽

Dominant Negative Mutations ◽

Guanine Nucleotide Binding

Heterotrimeric guanine nucleotide-binding proteins (G proteins) consisting of alpha, beta, and gamma subunits mediate signalling between cell surface receptors and intracellular effectors in eukaryotic cells. To define signalling functions of G gamma subunits (STE18 gene product) involved in pheromone response and mating in the yeast Saccharomyces cerevisiae, we isolated and characterized dominant-negative STE18 alleles. We obtained dominant-negative mutations that disrupt C-terminal sequences required for prenylation of G gamma precursors (CAAX box) and that affect residues in the N-terminal half of Ste18p. Overexpression of mutant G gamma subunits in wild-type cells blocked signal transduction; this effect was suppressed upon overexpression of G beta subunits. Mutant G gamma subunits may therefore sequester G beta subunits into nonproductive G beta gamma dimers. Because mutant G gamma subunits blocked the constitutive signal resulting from disruption of the G alpha subunit gene (GPA1), they are defective in functions required for downstream signalling. Ste18p bearing a C107Y substitution in the CAAX box displayed reduced electrophoretic mobility, consistent with a prenylation defect. G gamma subunits carrying N-terminal substitutions had normal electrophoretic mobilities, suggesting that these proteins were prenylated. G gamma subunits bearing substitutions in their N-terminal region or C-terminal CAAX box (C107Y) supported receptor-G protein coupling in vitro, whereas C-terminal truncations caused partial defects in receptor coupling.

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Monoclonal antibodies specific for globin chains

Blood ◽

10.1182/blood.v61.3.530.530 ◽

1983 ◽

Vol 61 (3) ◽

pp. 530-539 ◽

Cited By ~ 26

Author(s):

G Stamatoyannopoulos ◽

M Farquhar ◽

D Lindsley ◽

M Brice ◽

T Papayannopoulou ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Fetal Hemoglobin ◽

Beta Subunits ◽

Gamma Chain ◽

Globin Chains ◽

Gamma Subunits ◽

Species Specific ◽

Specific Reactions

Abstract Six monoclonal antibodies specific for human globin chains are described. They are produced by stable clones obtained by raising hybridomas using cells of mice immunized with either adult or fetal hemoglobin. Characterization of the antibodies included testing against tetrameric human and other animal hemoglobins, isolated hemoglobin chains, and when indicated, cyanogen bromide fragments. Monoclonals 16- 2 and 37–8 are beta-chain specific. Antibody 31–2 recognizes an antigenic determinant common to the alpha and beta subunits. Monoclonal 30–3 recognizes determinants best expressed in the alpha 2 beta 2 tetramer. Antibody 45–1 recognizes a determinant common to beta and gamma subunits, while antibody 51–7 is gamma-chain specific. None of the monoclonal antibodies recognizes mouse hemoglobin, and they display significant differences in binding to hemoglobins of various species. The species-specific reactions and the knowledge of the primary structures of globins allowed deductions about the antigenic sites recognized by two of the monoclonals (16–2 and 45–1). These antihemoglobin monoclonal antibodies will provide useful probes for studying hemoglobin expression in vivo and in vitro.

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Association of the gamma12 subunit of G proteins with actin filaments

Journal of Cell Science ◽

10.1242/jcs.110.13.1503 ◽

1997 ◽

Vol 110 (13) ◽

pp. 1503-1511

Author(s):

H. Ueda ◽

S. Saga ◽

H. Shinohara ◽

R. Morishita ◽

K. Kato ◽

...

Keyword(s):

G Proteins ◽

Actin Filaments ◽

Calf Serum ◽

C6 Glioma Cells ◽

Heterotrimeric G Proteins ◽

Differential Distribution ◽

Functional Differences ◽

Gamma Subunits ◽

Triton X

Recent studies have suggested an association between heterotrimeric G proteins, which play a major role in transmembrane signal transduction, and intracellular components. We therefore examined the subcellular localization of isoforms of G protein gamma subunits in Swiss 3T3 and C6 glioma cells, mainly containing the gamma5 and gamma12 subunits. Immunocytochemical double staining with phalloidin showed co-localization of the gamma12 subunit with actin filaments (F-actin), while the gamma5 co-localized with vinculin, suggesting an association with focal adhesion. Pretreatment of cells with Triton X-100 eliminated the gamma5 but not the gamma12 staining. Co-localization of gamma12 and F-actin was preserved when F-actin was disorganized with cytochalasin D or reorganized using fetal calf serum. Large amounts of gamma12 were recovered in the vimentin- and tubulin-free F-actin-rich fraction prepared from crude cytoskeleton preparations by double depolymerization-repolymerization. Co-localization of Gi2alpha, beta and gamma12 in the F-actin-rich fraction suggested the existence of gamma12 as a betagamma or heterotrimeric complex. Furthermore, purified betagamma12 was found to associate with F-actin in vitro more tightly than betagamma5. These results strongly suggest that the gamma12 subunit associates with F-actin in cells. The observed differential distribution of gamma12 and gamma5 implies functional differences for the two gamma subunits.

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Sequence and regulation of morphological and molecular events during the first cell cycle of mouse embryogenesis

Development ◽

10.1242/dev.87.1.175 ◽

1985 ◽

Vol 87 (1) ◽

pp. 175-206

Author(s):

Sarah K. Howlett ◽

Virginia N. Bolton

Keyword(s):

Cell Cycle ◽

Germinal Vesicle ◽

In Vitro Translation ◽

Careful Study ◽

Germinal Vesicle Breakdown ◽

Post Translational Modifications ◽

Molecular Events ◽

Terminal Events ◽

Sperm Penetration

Mouse oocytes were fertilized in vitro and the precise timing and sequence of morphological and molecular events occurring during the first cell cycle were investigated. The timing of development through the first cell cycle was found to be initiated by an event associated with sperm penetration rather than with germinal vesicle breakdown. DNA replication is initiated randomly in either pronucleus of a given egg, beginning approximately 11 h post insemination (hpi), and S phase lasting 6–7 h in both. Careful study of polypeptide synthetic profiles revealed three classes of changes in polypeptide synthesis during the first few hours of development: fertilization-independent, fertilization-accelerated, and fertilization-dependent. Pulse-chase experiments and in vitro translation of extracted mRNA showed that the changes in polypeptide synthetic profile result from differential mRNA activation, differential polypeptide turnover and post-translational modifications. These results support the notion that following ovulation, development is controlled at two levels. An endogenous (oocyte) programme, set in train by the terminal events of oocyte maturation, may regulate the ‘housekeeping’ functions of the egg, while sperm penetration activates a further endogenous (fertilization) programme, which may serve to initiate subsequent embryogenesis.

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Monoclonal antibodies specific for globin chains

Blood ◽

10.1182/blood.v61.3.530.bloodjournal613530 ◽

1983 ◽

Vol 61 (3) ◽

pp. 530-539 ◽

Cited By ~ 1

Author(s):

G Stamatoyannopoulos ◽

M Farquhar ◽

D Lindsley ◽

M Brice ◽

T Papayannopoulou ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Fetal Hemoglobin ◽

Beta Subunits ◽

Gamma Chain ◽

Globin Chains ◽

Gamma Subunits ◽

Species Specific ◽

Specific Reactions

Six monoclonal antibodies specific for human globin chains are described. They are produced by stable clones obtained by raising hybridomas using cells of mice immunized with either adult or fetal hemoglobin. Characterization of the antibodies included testing against tetrameric human and other animal hemoglobins, isolated hemoglobin chains, and when indicated, cyanogen bromide fragments. Monoclonals 16- 2 and 37–8 are beta-chain specific. Antibody 31–2 recognizes an antigenic determinant common to the alpha and beta subunits. Monoclonal 30–3 recognizes determinants best expressed in the alpha 2 beta 2 tetramer. Antibody 45–1 recognizes a determinant common to beta and gamma subunits, while antibody 51–7 is gamma-chain specific. None of the monoclonal antibodies recognizes mouse hemoglobin, and they display significant differences in binding to hemoglobins of various species. The species-specific reactions and the knowledge of the primary structures of globins allowed deductions about the antigenic sites recognized by two of the monoclonals (16–2 and 45–1). These antihemoglobin monoclonal antibodies will provide useful probes for studying hemoglobin expression in vivo and in vitro.

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