scholarly journals Light-dependent GTP-binding proteins in squid photoreceptors

1990 ◽  
Vol 272 (1) ◽  
pp. 79-85 ◽  
Author(s):  
P R Robinson ◽  
S F Wood ◽  
E Z Szuts ◽  
A Fein ◽  
H E Hamm ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
G Protein ◽  
G Proteins ◽  
Alpha Subunit ◽  
Protein G ◽  
Adp Ribosylation ◽  
Alpha Subunits ◽  
Gtp Binding ◽  
Molecular Masses ◽  
Lines Of Evidence

Previous biochemical and electrophysiological evidence suggests that in invertebrate photoreceptors, a GTP-binding protein (G-protein) mediates the actions of photoactivated rhodopsin in the initial stages of transduction. We find that squid photoreceptors contain more than one protein (molecular masses 38, 42 and 46 kDa) whose ADP-ribosylation by bacterial exotoxins is light-sensitive. Several lines of evidence suggest that these proteins represent distinct alpha subunits of G-proteins. (1) Pertussis toxin and cholera toxin react with distinct subsets of these polypeptides. (2) Only the 42 kDa protein immunoreacts with the monoclonal antibody 4A, raised against the alpha subunit of the G-protein of vertebrate rods [Hamm & Bownds (1984) J. Gen. Physiol. 84. 265-280]. (3) In terms of ADP-ribosylation, the 42 kDa protein is the least labile to freezing. (4) Of the 38 kDa and 42 kDa proteins, the former is preferentially extracted with hypo-osmotic solutions, as demonstrated by the solubility of its ADP-ribosylated state and by the solubility of the light-dependent binding of guanosine 5′-[gamma-thio]triphosphate. The specific target enzymes for the observed G-proteins have not been established.

scholarly journals G-proteins in skeletal muscle. Evidence for a 40 kDa pertussis-toxin substrate in purified transverse tubules

1988 ◽  
Vol 254 (2) ◽  
pp. 405-409 ◽  
Author(s):  
M Toutant ◽  
J Barhanin ◽  
J Bockaert ◽  
B Rouot
Keyword(s):  
Skeletal Muscle ◽  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Excitation Contraction Coupling ◽  
Contraction Coupling ◽  
Adp Ribosylation ◽  
Alpha Subunits ◽  
Low Salt ◽  
T Tubules

In muscle, it has been established that guanosine 5′-[gamma-thio]triphosphate (GTP[S]), a non-hydrolysable GTP analogue, elicits a rise in tension in chemically skinned fibres, and that pretreatment with Bordetella pertussis toxin (PTX) decreases GTP[S]-induced tension development [Di Virgilio, Salviati, Pozzan & Volpe (1986) EMBO J. 5, 259-262]. In the present study, G-proteins were analysed by PTX-catalysed ADP-ribosylation and by immunoblotting experiments at cellular and subcellular levels. First, the nature of the G-proteins present in neural and aneural zones of rat diaphragm muscle was investigated. PTX, known to catalyse the ADP-ribosylation of the alpha subunit of several G-proteins, was used to detect G-proteins. Three sequential extractions (low-salt-soluble, detergent-soluble and high-salt-soluble) were performed, and PTX was found to label two substrates of 41 and 40 kDa only in the detergent-soluble fraction. The addition of pure beta gamma subunits of G-proteins to the low-salt-soluble extract did not provide a way to detect PTX-catalysed ADP-ribosylation of G-protein alpha subunits in this hydrophilic fraction. In neural as well as in aneural zones, the 39 kDa PTX substrate, very abundant in the nervous system (Go alpha), was not observed. We then studied the nature of the G alpha subunits present in membranes from transverse tubules (T-tubules) purified from rabbit skeletal muscle. Only one 40 kDa PTX substrate was found in T-tubules, known to be the key element of excitation-contraction coupling. The presence of a G-protein in T-tubule membranes was further confirmed by the immunoreactivity detected with an anti-beta-subunit antiserum. A 40 kDa protein was also detected in T-tubule membranes with an antiserum raised against a purified bovine brain Go alpha. The presence of two PTX substrates (41 and 40 kDa) in equal amounts in total muscle extracts, compared with only one (40 kDa) found in purified T-tubule membranes, suggests that this 40 kDa PTX substrate might be involved in excitation-contraction coupling.

scholarly journals Homologous and unique G protein alpha subunits in the nematode Caenorhabditis elegans.

1991 ◽  
Vol 2 (2) ◽  
pp. 135-154 ◽  
Author(s):  
M A Lochrie ◽  
J E Mendel ◽  
P W Sternberg ◽  
M I Simon
Keyword(s):  
Amino Acid ◽  
G Protein ◽  
G Proteins ◽  
Amino Acid Sequences ◽  
Alpha Subunit ◽  
Predicted Amino Acid Sequence ◽  
C Elegans ◽  
Alpha Subunits ◽  
G Protein Alpha Subunit ◽  
Protein Alpha Subunit

A cDNA corresponding to a known G protein alpha subunit, the alpha subunit of Go (Go alpha), was isolated and sequenced. The predicted amino acid sequence of C. elegans Go alpha is 80-87% identical to other Go alpha sequences. An mRNA that hybridizes to the C. elegans Go alpha cDNA can be detected on Northern blots. A C. elegans protein that crossreacts with antibovine Go alpha antibody can be detected on immunoblots. A cosmid clone containing the C. elegans Go alpha gene (goa-1) was isolated and mapped to chromosome I. The genomic fragments of three other C. elegans G protein alpha subunit genes (gpa-1, gpa-2, and gpa-3) have been isolated using the polymerase chain reaction. The corresponding cosmid clones were isolated and mapped to disperse locations on chromosome V. The sequences of two of the genes, gpa-1 and gpa-3, were determined. The predicted amino acid sequences of gpa-1 and gpa-3 are only 48% identical to each other. Therefore, they are likely to have distinct functions. In addition they are not homologous enough to G protein alpha subunits in other organisms to be classified. Thus C. elegans has G proteins that are identifiable homologues of mammalian G proteins as well as G proteins that appear to be unique to C. elegans. Study of identifiable G proteins in C. elegans may result in a further understanding of their function in other organisms, whereas study of the novel G proteins may provide an understanding of unique aspects of nematode physiology.

Disturbing Gtp-Binding Protein Function Through Microinjection Into The Visual Cell Of Limulus

1992 ◽  
Vol 47 (11-12) ◽  
pp. 915-921 ◽  
Author(s):  
Henmg Stieve ◽  
Barbara Niemeyer ◽  
Klaus Aktories ◽  
Heidi E. Hamm
Keyword(s):  
Monoclonal Antibody ◽  
G Protein ◽  
Protein Function ◽  
Clostridium Botulinum ◽  
Functional Role ◽  
Electrical Response ◽  
Visual Cell ◽  
Gtp Binding ◽  
Dim Light ◽  
Ventral Nerve Photoreceptor

We have tested the action of three agents microinjected into the ventral nerve photoreceptor of Limulus on the electrical response to dim light. 1. A monoclonal antibody (mAb 4 A) against the Gɑ subunit of frog transducin reduces the size of the receptor current to 60%, suggesting an interaction with Gɑ in the Limulus photoreceptor. 2. Injection of Clostridium botulinum ADPribosyltransferase C 3 reduces the size to 46%; latency is not affected. The results imply that small GTP-binding proteins play a functional role in photoreception of invertebrates. 3. Injection of GD P-β-S reduces dose-dependently the size of the receptor current to 15% and prolongs the latency to 200%, presumably by reducing number and rate of G-protein activations

scholarly journals Erratum

1991 ◽  
Vol 11 (4) ◽  
pp. 706-706
Keyword(s):  
Blood Flow ◽  
Cerebral Blood Flow ◽  
Equilibrium State ◽  
Rat Brain ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Adp Ribosylation ◽  
Gtp Binding ◽  
Reconstituted System ◽  
Α Subunits

Ischemia of Rat Brain Decreases Pertussis Toxin-Catalyzed [32P] ADP Ribosylation of GTP-Binding Proteins (Gi1 and G0) in Membranes Katsunobu Takenaka, Yasunori Kanaho, Koh-ichi Nagata, Noboru Sakai, Hiromu Yamada, Yoshinori Nozawa [ Originally published in Journal of Cerebral Blood Flow and Metabolism 1991;11:155–160] On page 158 of the above, arrows were erroneously deleted from the equation in the following passage: Heterotrimers of G proteins that bind GDP to α subunits seem to be the preferred substrates for PTcatalyzed ADP ribosylation since guanine nucleotides (GDP and GTP) and 13'Y subunits stimulate ADP ribosylation in the reconstituted system and in membranes (Tsai et aI., 1984). These results indicate that the G proteins may exist at the equilibrium state as shown below: This omission was the result of a typesetting error, which the publisher regrets.

Mutated alpha subunit of the Gq protein induces malignant transformation in NIH 3T3 cells

1992 ◽  
Vol 12 (10) ◽  
pp. 4687-4693
Author(s):  
G Kalinec ◽  
A J Nazarali ◽  
S Hermouet ◽  
N Xu ◽  
J S Gutkind
Keyword(s):  
Adenylyl Cyclase ◽  
G Proteins ◽  
Alpha Subunit ◽  
Endocrine Tumors ◽  
3T3 Cells ◽  
Activating Mutations ◽  
Alpha Subunits ◽  
G Alpha Q ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

The discovery of mutated, GTPase-deficient alpha subunits of Gs or Gi2 in certain human endocrine tumors has suggested that heterotrimeric G proteins play a role in the oncogenic process. Expression of these altered forms of G alpha s or G alpha i2 proteins in rodent fibroblasts activates or inhibits endogenous adenylyl cyclase, respectively, and causes certain alterations in cell growth. However, it is not clear whether growth abnormalities result from altered cyclic AMP synthesis. In the present study, we asked whether a recently discovered family of G proteins, Gq, which does not affect adenylyl cyclase activity, but instead mediates the activation of phosphatidylinositol-specific phospholipase C harbors transforming potential. We mutated the cDNA for the alpha subunit of murine Gq in codons corresponding to a region involved in binding and hydrolysis of GTP. Similar mutations unmask the transforming potential of p21ras or activate the alpha subunits of Gs or Gi2. Our results show that when expressed in NIH 3T3 cells, activating mutations convert G alpha q into a dominant acting oncogene.

G-proteins in mammalian gametes: an immunocytochemical study

1988 ◽  
Vol 91 (1) ◽  
pp. 21-31
Author(s):  
N.B. Garty ◽  
D. Galiani ◽  
A. Aharonheim ◽  
Y.K. Ho ◽  
D.M. Phillips ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
G Protein ◽  
G Proteins ◽  
Cumulus Cells ◽  
Bovine Brain ◽  
Regulatory Proteins ◽  
Immunocytochemical Study ◽  
Cell Cytoplasm ◽  
Sperm Tail ◽  
Immunoreactive Material

The presence of transductory GTP(G)-regulatory proteins in mammalian gametes has been examined by indirect fluorescence immunocytochemistry. Using rabbit antisera to bovine rod beta gamma-transducin (RA beta gamma T), bovine rod holotransducin (AS-1), bovine rod alpha-transducin (RA alpha T), synthetic bovine rod alpha-transducin C-terminal decapeptide (AS-6), bovine brain alpha 39Go (RA alpha 39), and two mouse monoclonal antibodies raised against frog retinal transducin (4A), and rat brain beta-tubulin, we demonstrated the presence of corresponding immunoreactive material in both rat oocytes and bovine ejaculated sperm. Immunostaining in the oocyte was evenly distributed on the oolemma, excluding the cell cytoplasm and zona pellucida. Immunoreactive material was also present in the cumulus cells that encapsulate the oocyte. In contrast, the immunofluorescence corresponding to transductory G-proteins was confined in sperm to functionally defined regions in the head and tail, in a manner specific for each antibody. While RA beta gamma T, AS-1 and RA alpha 39 all stained the entire acrosome, AS-6 and RA alpha T stained only the acrosomal tip. Monoclonal antibody 4A stained the midpiece exclusively and anti-rat betaq-tubulin (a structural G-protein) stained the full length of the sperm tail. The existence of several G-protein types in mammalian gametes suggests their possible involvement in the regulation of various effector systems, in a manner reminiscent of somatic cells. The unique situation in sperm, where different G-proteins show distinct and specific patterns of distribution, further suggests their association with various effector systems in discrete functional domains.

scholarly journals The carboxy-terminal domain of Gs alpha is necessary for anchorage of the activated form in the plasma membrane.

1990 ◽  
Vol 111 (4) ◽  
pp. 1427-1435 ◽  
Author(s):  
Y Audigier ◽  
L Journot ◽  
C Pantaloni ◽  
J Bockaert
Keyword(s):  
Adenylate Cyclase ◽  
Binding Proteins ◽  
Alpha Subunit ◽  
Amino Terminus ◽  
Gtp Binding Proteins ◽  
Amino Terminal ◽  
Alpha Subunits ◽  
Gtp Binding ◽  
Carboxy Terminal ◽  
Gs Alpha

GTP-binding proteins which participate in signal transduction share a common heterotrimeric structure of the alpha beta gamma-type. In the activated state, the alpha subunit dissociates from the beta gamma complex but remains anchored in the membrane. The alpha subunits of several GTP-binding proteins, such as Go and Gi, are myristoylated at the amino terminus (Buss, J. E., S. M. Mumby, P. J. Casey, A. G. Gilman, and B. M. Sefton. 1987. Proc. Natl. Acad. Sci. USA. 84:7493-7497). This hydrophobic modification is crucial for their membrane attachment. The absence of fatty acid on the alpha subunit of Gs (Gs alpha), the protein involved in adenylate cyclase activation, suggests a different mode of anchorage. To characterize the anchoring domain of Gs alpha, we used a reconstitution model in which posttranslational addition of in vitro-translated Gs alpha to cyc- membranes (obtained from a mutant of S49 cell line which does not express Gs alpha) restores the coupling between the beta-adrenergic receptor and adenylate cyclase. The consequence of deletions generated by proteolytic removal of amino acid sequences or introduced by genetic removal of coding sequences was determined by analyzing membrane association of the proteolyzed or mutated alpha chains. Proteolytic removal of a 9-kD amino-terminal domain or genetic deletion of 28 amino-terminal amino acids did not modify the anchorage of Gs alpha whereas proteolytic removal of a 1-kD carboxyterminal domain abolished membrane interaction. Thus, in contrast to the myristoylated alpha subunits which are tethered through their amino terminus, the carboxy-terminal residues of Gs alpha are required for association of this protein with the membrane.

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