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 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 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.

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.

scholarly journals Determination of interaction sites of phospholipase D1 for RhoA

2001 ◽  
Vol 355 (3) ◽  
pp. 779-785 ◽  
Author(s):  
Songmin CAI ◽  
John H. EXTON
Keyword(s):  
Dependent Manner ◽  
Interaction Site ◽  
Interaction Sites ◽  
Kinase C ◽  
C Terminus ◽  
Phospholipase D1 ◽  
A Site ◽  
Adp Ribosylation Factor ◽  
Made In

Phospholipase D (PLD) is regulated by many factors, including protein kinase C (PKC) and small G-proteins of the Rho and ADP-ribosylation factor families. Previous studies revealed that the interaction site of human PLD1 for RhoA is located in its C-terminus, but the exact locus has not been determined. The purpose of the present study was to determine the interaction site of rat PLD1 (rPLD1) with RhoA. Selection with phage display of different peptides of rPLD1 confirmed that GTP-bound RhoA interacted with a site in the amino acid sequence 873–1024 at the C-terminus of rPLD1. RhoA also associated with this peptide in a GTP-dependent manner in COS-7 cell lysates and the peptide inhibited RhoA stimulation of PLD activity in membranes from COS-7 cells expressing rPLD1. A series of alanine mutations of non-conserved residues were made in this sequence, and the enzymes were expressed in COS-7 cells and checked for responses to activation of PKC, which interacts with the N-terminus of PLD1, and also to the constitutively active V14RhoA. Mutations in the C-terminus of rPLD1 (K946A, V950A, R955A and K962A) caused partial loss of V14RhoA stimulation, and double mutations (K946A/K962A, K946A/V950A and K962A/V950A) caused an almost total loss. Co-immunoprecipitation studies also showed that the mutated forms of rPLD1 described above failed to bind V14RhoA compared with wild-type rPLD1, whereas rPLD1 with mutations outside the region K946–K962 bound V14RhoA normally. It is concluded that basic amino acids in a restricted C-terminal region of rPLD1 are important for binding of RhoA and its activation of PLD activity.

scholarly journals 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.

scholarly journals GTP analogues promote release of the α subunit of the guanine nucleotide binding protein, Gi2, from membranes of rat glioma C6 BU1 cells

1988 ◽  
Vol 254 (2) ◽  
pp. 391-396 ◽  
Author(s):  
G Milligan ◽  
I Mullaney ◽  
C G Unson ◽  
L Marshall ◽  
A M Spiegel ◽  
...  
Keyword(s):  
G Protein ◽  
Pertussis Toxin ◽  
Nucleotide Binding ◽  
Alpha Subunit ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding Protein ◽  
Rat Glioma ◽  
Glioma C6 ◽  
Gamma Subunits ◽  
Guanine Nucleotide Binding

The major pertussis-toxin-sensitive guanine nucleotide-binding protein of rat glioma C6 BU1 cells corresponded immunologically to Gi2. Antibodies which recognize the alpha subunit of this protein indicated that it has an apparent molecular mass of 40 kDa and a pI of 5.7. Incubation of membranes of these cells with guanosine 5′-[beta gamma-imido]triphosphate, or other analogues of GTP, caused release of this polypeptide from the membrane in a time-dependent manner. Analogues of GDP or of ATP did not mimic this effect. The GTP analogues similarly caused release of the alpha subunit of Gi2 from membranes of C6 cells in which this G-protein had been inactivated by pretreatment with pertussis toxin. The beta subunit was not released from the membrane under any of these conditions, indicating that the release process was a specific response to the dissociation of the G-protein after binding of the GTP analogue. Similar nucleotide profiles for release of the alpha subunits of forms of Gi were noted for membranes of both the neuroblastoma x glioma hybrid cell line NG108-15 and of human platelets. These data provide evidence that: (1) pertussis-toxin-sensitive G-proteins, in native membranes, do indeed dissociate into alpha and beta gamma subunits upon activation; (2) the alpha subunit of ‘Gi-like’ proteins need not always remain in intimate association with the plasma membrane; and (3) the alpha subunit of Gi2 can still dissociate from the beta/gamma subunits after pertussis-toxin-catalysed ADP-ribosylation.

scholarly journals Binding of the γ-subunit of retinal rod-outer-segment phosphodiesterase with both transducin and the catalytic subunits of phosphodiesterase

1990 ◽  
Vol 271 (3) ◽  
pp. 721-727 ◽  
Author(s):  
J M Cunnick ◽  
D Hurt ◽  
B Oppert ◽  
K Sakamoto ◽  
D J Takemoto
Keyword(s):  
Outer Segment ◽  
Carboxypeptidase Y ◽  
Guanine Nucleotide Binding Protein ◽  
Catalytic Subunits ◽  
Multifunctional Protein ◽  
Rod Outer Segment ◽  
C Terminus ◽  
Retinal Rod ◽  
Alpha Beta

The gamma-subunit of retinal rod-outer-segment phosphodiesterase (PDE-gamma) is a multifunctional protein which interacts directly with both of the catalytic subunits of PDE (PDE alpha/beta) and the alpha-subunit of the retinal G (guanine-nucleotide-binding)-protein transducin alpha (T alpha). We have previously reported that the PDE gamma binds to T alpha at residue nos. 24-45 [Morrison. Rider & Takemoto (1987) FEBS Lett. 222, 266-270]. In vitro this results in inhibition of T alpha GTP/GDP exchange [Morrison, Cunnick, Oppert & Takemoto (1989) J. Biol. Chem. 264, 11671-11681]. We now report that the inhibitory region of PDE gamma for PDE alpha/beta occurs at PDE gamma residues 54-87. This binding results in inhibition of either trypsin-solubilized or membrane-bound PDE alpha/beta. PDE gamma which has been treated with carboxypeptidase Y, removing the C-terminus, does not inhibit PDE alpha/beta, but does inhibit T alpha GTP/GDP exchange. Inhibition by PDE gamma can be removed by T alpha-guanosine 5′-[gamma-thio]triphosphate (GTP[S]) addition to membranes. This results in a displacement of PDE gamma, but not in removal of this subunit from the membrane [Whalen, Bitensky & Takemoto (1990) Biochem. J. 265, 655-658]. These results suggest that low levels of T alpha-GTP[S] can result in displacement of PDE gamma from the membrane in vitro as a GTP[S]-T alpha-PDE gamma complex. Further activation by high levels of T alpha-GTP[S] occurs by displacement of PDE gamma from its inhibitory site on PDE alpha/beta, but not in removal from the membrane.

Sign in / Sign up

Export Citation Format

Share Document