scholarly journals Regulation of retinal transducin by C-terminal peptides of rhodopsin

1985 ◽  
Vol 232 (3) ◽  
pp. 669-672 ◽  
Author(s):  
D J Takemoto ◽  
L J Takemoto ◽  
J Hansen ◽  
D Morrison
Keyword(s):  
Gtpase Activity ◽  
Amino Acid Residues ◽  
Guanine Nucleotide Binding Protein ◽  
Gamma Subunit ◽  
Internal Loop ◽  
External Loop ◽  
Loop Region ◽  
C Terminus ◽  
Retinal Rod ◽  
Guanine Nucleotide Binding

Transducin is a multi-subunit guanine-nucleotide-binding protein that mediates signal coupling between rhodopsin and cyclic GMP phosphodiesterase in retinal rod outer segments. Whereas the T alpha subunit of transducin binds guanine nucleotides and is the activator of the phosphodiesterase, the T beta gamma subunit may function to link physically T alpha with photolysed rhodopsin. In order to determine the binding sites of rhodopsin to transducin, we have synthesized eight peptides (Rhod-1 etc.) that correspond to the C-terminal regions of rhodopsin and to several external and one internal loop region. These peptides were tested for their inhibition of restored GTPase activity of purified transducin reconstituted into depleted rod-outer-segment disc membranes. A marked inhibition of GTPase activity was observed when transducin was pre-incubated with peptides Rhod-1, Rhod-2 and Rhod-3. These peptides correspond to opsin amino acid residues 332-339, 324-331 and 317-321 respectively. Peptides corresponding to the three external loop regions or to the C-terminal residues 341-348 did not inhibit reconsituted GTPase activity. Likewise, Rhod-8, a peptide corresponding to an internal loop region of rhodopsin, did not inhibit GTPase activity. These findings support the concept that these specific regions of the C-terminus of rhodopsin serve as recognition sites for transducin.

scholarly journals C-terminal peptides of rhodopsin. Determination of the optimum sequence for recognition of retinal transducin

1986 ◽  
Vol 235 (1) ◽  
pp. 309-312 ◽  
Author(s):  
D J Takemoto ◽  
D Morrison ◽  
L C Davis ◽  
L J Takemoto
Keyword(s):  
Amino Acid Residues ◽  
Guanine Nucleotide Binding Protein ◽  
Bovine Rhodopsin ◽  
C Terminus ◽  
Retinal Rod ◽  
Gamma Subunits ◽  
Guanine Nucleotide Binding ◽  
A Site ◽  
Optimum Sequence

In vertebrate retinal rod outer segments, transducin, a guanine-nucleotide-binding protein, mediates signal coupling between rhodopsin and cyclic GMP phosphodiesterase. Whereas the T alpha subunit (39 kDa) of transducin binds guanine nucleotides and is the activator of the phosphodiesterase, the T beta gamma subunits (35 and 10 kDa) may function to physically link T alpha with photolysed rhodopsin. We have previously reported that a site of binding of transducin is on the C-terminus of bovine rhodopsin. By using competition with synthetic peptides, the recognition region was localized to bovine opsin amino acid residues 317-339. Further studies are detailed which determine the boundaries of this binding site on rhodopsin, as well as some of the critical amino acids needed for transducin binding. These results suggest that the serine and threonine residues in the rhodopsin C-terminal peptides Rhod-1 and Rhod-3 are critical for reconstitution of transducin GTPase activity.

scholarly journals Foetal-calf serum stimulates a pertussis-toxin-sensitive high-affinity GTPase activity in rat glioma C6 BU1 cells

1987 ◽  
Vol 245 (2) ◽  
pp. 501-505 ◽  
Author(s):  
G Milligan
Keyword(s):  
Pertussis Toxin ◽  
Cellular Proliferation ◽  
Calf Serum ◽  
Foetal Calf Serum ◽  
Gtpase Activity ◽  
Guanine Nucleotide Binding Protein ◽  
Rat Glioma ◽  
High Affinity ◽  
Glioma C6 ◽  
Guanine Nucleotide Binding

Cellular proliferation of rat glioma C6 BU1 cells in tissue culture is dependent on the presence of either calf or foetal-calf serum in the medium. Foetal-calf serum stimulated a high-affinity GTPase in membranes derived from C6 BU1 cells. Pretreatment of the cells with pertussis toxin decreased the high-affinity GTPase activity substantially, and attenuated the foetal-calf-serum-stimulated increase in this GTPase activity. Cholera toxin, in contrast, did not modulate the response to foetal-calf serum. Foetal-calf serum did not inhibit adenylate cyclase activity in membranes of these cells, indicating that the G-protein that was stimulated by foetal-calf serum was not Gi (the inhibitory one). Although the nature of the specific component of foetal-calf serum responsible for this pertussis-toxin-sensitive receptor-mediated stimulation of high-affinity GTPase activity has not been identified, it was mimicked neither by bombesin, which can stimulate inositol phospholipid turnover via a guanine nucleotide binding protein, nor by platelet-derived growth factor, which is present in substantial concentrations in foetal-calf serum. This report represents the first demonstration of a pertussis-toxin-substrate-mediated response in this cell line and provides further evidence that G-proteins other than Gi can be functionally inactivated by pertussis toxin.

scholarly journals Evidence that activation of a common G-protein by receptors for leukotriene B4 and N-formylmethionyl-leucyl-phenylalanine in HL-60 cells occurs by different mechanisms

1989 ◽  
Vol 260 (2) ◽  
pp. 427-434 ◽  
Author(s):  
K R McLeish ◽  
P Gierschik ◽  
T Schepers ◽  
D Sidiropoulos ◽  
K H Jakobs
Keyword(s):  
Cholera Toxin ◽  
G Protein ◽  
Leukotriene B4 ◽  
Alpha Subunit ◽  
Gtpase Activity ◽  
Protein Activity ◽  
Guanine Nucleotide Binding Protein ◽  
Gtp Hydrolysis ◽  
Fmlp Receptor ◽  
Guanine Nucleotide Binding

Differentiated HL-60 cells were found to respond to the chemoattractants leukotriene B4 (LTB4) and N-formylmethionyl-leucyl-phenylalanine (FMLP), in a manner similar to neutrophils. Membranes of myeloid differentiated HL-60 cells were used (a) to examine the ability of LTB4 receptors to interact with a guanine-nucleotide-binding protein (G-protein), and (b) to compare this G-protein with that which is coupled to the FMLP receptor. LTB4 stimulated a dose-dependent increase in GTP hydrolysis and guanosine 5′-[gamma-thio]triphosphate (GTP[S]) binding, demonstrating that LTB4 receptors on HL-60 cells are coupled to a G-protein. Both pertussis toxin and cholera toxin inhibited stimulation of GTPase activity and GTP[S] binding by either LTB4 or FMLP, indicating that both receptors are coupled to a G-protein containing a 40 kDa alpha-subunit. That the two receptors share a common G-protein was shown by FMLP enhancement of cholera-toxin-induced inhibition of GTPase activity stimulated by either FMLP or LTB4. However, LTB4 did not enhance cholera-toxin-induced inhibition of GTPase activity, suggesting that the receptors interacted differently with this G-protein. This difference was confirmed by showing that FMLP, but not LTB4, stimulated receptor-specific [32P]ADP-ribosylation of the 40 kDa alpha-subunit. Concentrations of LTB4 and FMLP which produced maximal responses produced enhanced stimulation in both assays. This additive effect was not abolished by inactivation of up to 80% of G-protein activity by N-ethylmaleimide or cholera toxin. We conclude that LTB4 and FMLP receptors in HL-60 cells are coupled to a common G-protein. The receptor-G-protein interaction is different for the two receptors, and G-proteins not coupled to both receptors may account for the additive response.

scholarly journals Molecular cloning and primary structure of squid (Loligo forbesi) rhodopsin, a phospholipase C-directed G-protein-linked receptor

1991 ◽  
Vol 274 (1) ◽  
pp. 35-40 ◽  
Author(s):  
M D Hall ◽  
M A Hoon ◽  
N J P Ryba ◽  
J D D Pottinger ◽  
J N Keen ◽  
...  
Keyword(s):  
G Protein ◽  
Primary Structure ◽  
Protein G ◽  
Guanine Nucleotide ◽  
Cdna Sequencing ◽  
Functional Importance ◽  
Guanine Nucleotide Binding Protein ◽  
C Terminus ◽  
Loligo Forbesi ◽  
Guanine Nucleotide Binding

The sequence of squid (Loligo forbesi) rhodopsin was determined by protein and cDNA sequencing. The protein has close similarity to octopus rhodopsin, having an N-terminal region (residues 1-340) which resembles other guanine-nucleotide-binding protein (G-protein)-linked receptors and a repetitive proline-rich C-terminus (residues 340-452). Comparison of the sequence of squid rhodopsin with those of other members of the G-protein-linked receptor superfamily reveals features which we predict to have both structural and functional importance.

scholarly journals Inhibition of the GTPase activity of transducin by an NAD+:arginine ADP-ribosyltransferase from turkey erythrocytes

1987 ◽  
Vol 248 (3) ◽  
pp. 749-754 ◽  
Author(s):  
P A Watkins ◽  
Y Kanaho ◽  
J Moss
Keyword(s):  
Nucleotide Binding ◽  
Rod Outer Segments ◽  
Guanine Nucleotide ◽  
Gtpase Activity ◽  
Adp Ribosylation ◽  
Outer Segments ◽  
Retinal Rod ◽  
Guanine Nucleotide Binding ◽  
Adp Ribosyltransferase ◽  
Retinal Rod Outer Segments

The bacterial toxins, choleragen and pertussis toxin, inhibit the light-stimulated GTPase activity of bovine retinal rod outer segments by catalysing the ADP-ribosylation of the alpha-subunit (T alpha) of transducin [Abood, Hurley, Pappone, Bourne & Stryer (1982) J. Biol. Chem. 257, 10540-10543; Van Dop, Yamanaka, Steinberg, Sekura, Manclark, Stryer & Bourne (1984) J. Biol. Chem. 259, 23-26]. Incubation of retinal rod outer segments with NAD+ and a purified NAD+:arginine ADP-ribosyltransferase from turkey erythrocytes resulted in approx. 60% inhibition of GTPase activity. Inhibition was dependent on both enzyme and NAD+, and was potentiated by the non-hydrolysable GTP analogues guanosine 5′-[beta gamma-imido]triphosphate (p[NH]ppG) and guanosine 5′-[beta gamma-methylene]triphosphate (p[CH2]ppG). The transferase ADP-ribosylated both the T alpha and T beta subunits of purified transducin. T alpha (39 kDa), after ADP-ribosylation, migrated as two distinct peptides with molecular masses of 42 kDa and 46 kDa on SDS/polyacrylamide-gel electrophoresis. T beta (36 kDa), after ADP-ribosylation, migrated as a 38 kDa peptide. With purified transducin subunits, it was observed that the GTPase activity of ADP-ribosylated T alpha, reconstituted with unmodified T beta gamma and photolysed rhodopsin, was decreased by 80%; conversely, reconstitution of T alpha with ADP-ribosyl-T beta gamma resulted in only a 19% inhibition of GTPase. Thus ADP-ribosylation of T alpha, the transducin subunit that contains the guanine nucleotide-binding site, has more dramatic effects on GTPase activity than does modification of the critical ‘helper subunits’ T beta gamma. To elucidate the mechanism of GTPase inhibition by transferase, we studied the effect of ADP-ribosylation on p[NH]pp[3H]G binding to transducin. It was shown previously that modification of transducin by choleragen, which like transferase ADP-ribosylates arginine residues, did not affect guanine nucleotide binding. ADP-ribosylation by the transferase, however, decreased p[NH]pp[3H]G binding, consistent with the hypothesis that choleragen and transferase inhibit GTPase by different mechanisms.

The ras-like protein p25rab3A is partially cytosolic and is expressed only in neural tissue

1989 ◽  
Vol 9 (11) ◽  
pp. 4807-4811
Author(s):  
E Burstein ◽  
I G Macara
Keyword(s):  
Rat Brain ◽  
Binding Protein ◽  
Neural Tissue ◽  
Immunoblot Analysis ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding Protein ◽  
C Terminus ◽  
Number Of Genes ◽  
Membrane Bound ◽  
Guanine Nucleotide Binding

An antipeptide antiserum has been developed against a sequence near the C terminus of the small guanine nucleotide-binding protein p25rab3A. This protein is the product of one of a large number of genes that show homology to the ras proto-oncogenes. Immunoblotting with the antiserum specifically detected a 25-kilodalton protein in brain membranes. This protein coeluted from a MonoQ high-resolution ion-exchange column with a 25-kilodalton GTP-binding protein at a salt concentration similar to that known to elute purified p25rab3A. Unlike p21ras, which is exclusively membrane bound, p25rab3A is present in both the cytosol and membrane fractions of rat brain. It was not detected in other tissues, although a band of slightly lower molecular weight was observed with skeletal muscle. Western blot (immunoblot) analysis of five regions of the rat brain indicated that p25rab3A is most abundant in the hypothalamus and hippocampus.

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