Detection of 23-27 kDa GTP-binding proteins in platelets and other cells

Bhullar, Rajinder P.; Haslam, Richard J.

doi:10.1042/bj2450617

Cited by 167 publications

(69 citation statements)

References 35 publications

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“…As first reported by Bhullar and Haslam [21], the small G proteins, unlike the a-subunits of heterotrimeric G proteins, retain their ability to bind GTP after polyacrylamide gel electrophoresis and transfer to nitrocellulose membranes. We used this method to study whether bovine ROS membranes also contain such proteins.…”

Section: Resultsmentioning

confidence: 66%

Interaction of small G proteins with photoexcited rhodopsin

et al. 1990

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Bovine rod outer segment (ROS) membranes contain in addition to the heterotrimeric G protein transducin, several small GTP-binding proteins (23-27 kDa). Furthermore, these membranes contain two substrate proteins (about 22 and 24 kDa) for botulinurn C3 ADP-ribosyltransferase known to ADP-ribosylate small G proteins in any mammalian cell type studied so far. Most interestingly, P*P]ADP-ribosylation of ROS membrane small G proteins by C3 is regulated by light and guanine nucleotides in a manner similar to pertussis toxin-catalyzed[32P]ADP-ribosy1ation of the a-subunit of transducin.These findings suggest that not only the heterotrimeric G protein transducin but also the C3 substrate small G proteins present in ROS membranes interact with photoexcited rhodopsin and thus contribute to its signalling action.GTP-binding protein; Rhodopsin; Botulinum C3 ADP-ribosyl-transferase; Transducin; (Bovine rod outer segment)

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Section: Resultsmentioning

confidence: 66%

Interaction of small G proteins with photoexcited rhodopsin

et al. 1990

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“…To test the specificity of binding of [a-3ZP]GTP to blotted recombinant c-Ha-ras, we added an excess of unlabelled nucleotide to the binding reaction. Addition of 10 pM GTP or 10 pM GDP to the standard incubation abolished binding of [a-32P]GTP completely, whereas 10 pM ATP was less effective ( Fig. 2A).…”

Section: Resultsmentioning

confidence: 96%

GTP‐blot Analysis of Small GTP‐binding Proteins

Klinz

1994

European Journal of Biochemistry

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Recombinant c-Ha-ras, ralA and rap2, but not raplA or raplB proteins retained their ability to bind [a-32P]GTP after SDSPAGE and transfer to nitrocellulose. Recombinant c-Ha-ras missing the C-terminal 23 amino acid residues failed to bind [a-32P]GTP after the blot, and the ability of recombinant ralA missing the C-terminal 28 amino acid residues to bind [a-32P]GTP was decreased manyfold. The presence of nonionic detergents of the polyoxyethylene type such as Tween 20, Triton X-100, Nonidet P40 or Lubrol PX in the incubation buffer was necessary to induce renaturation of blotted recombinant c-Ha-ras protein, whereas other types of detergents were ineffective. We propose that detergents of the polyoxyethylene type induce the refolding of some types of blotted small GTP-binding proteins and that the C-terminus is involved in the refolding process.Membranes from NIH3T3 fibroblasts overexpressing c-Ha-ras protein showed much weaker binding of [a-32P]GTP as expected from the level of ras immunoreactivity. Treatment of fibroblasts with lovastatin, an inhibitor of hydroxymethylglutaryl-coenzyme A reductase, caused the accumulation of the unfarnesylated form of c-Ha-ras in the cytosol. Examination of [a-32P]GTP-binding and immunoreactivity for cytosolic and membrane-bound c-Ha-ras revealed that binding of [CX-~~P]GTP to unprocessed c-Ha-ras was increased about threefold compared to the same amount of processed c-Ha-ras.Our results demonstrate that detection and quantification of small GTP-binding proteins in eukaryotic cells by GTP-blot analysis is hampered by the fact that these proteins differ strongly in their ability to renature after blotting to nitrocellulose.Guanine nucleotide binding regulatory proteins such as ras proteins and a-subunits of heterotrimeric GTP-binding proteins exert their function by cycling between an inactive, GDP-bound, and an active, Members of the mammalian ras protooncogene group, c-Ha-ras, c-Ki-ras and N-ras each encode small guanine nucleotide binding proteins of 21 kDa which are associated with the inner side of the plasma membrane. Mammalian ras genes acquire transformation-inducing properties by single point mutations within their coding sequences [l, 31. Cloning and sequencing of cDNAs has revealed that a large and diverse family of ras-related small GTP-binding proteins exist with calculated molecular masses between 21 -25 kDa. Groups belonging to this family include the rap, ral, rho, rac and rab proteins [4,5]. Members of the family of small GTPbinding proteins show an extraordinary conservation of sequences dedicated to guanine nucleotide binding.To crystallize the human c-Ha-ras protooncogene product, John et al. [6] expressed the protein with a truncated Cterminus of 23 amino acids. The resulting c-Ha-ras (amino acid residues 1-166) was crystallized as a complex with the slowly hydrolyzing GTP analogue guanosine 5'- [P,yimido]triphosphate (GppNHp) and analyzed by X-ray crys-

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“…[ ␣-32 P]-GTP-blot Overlay Assay Low molecular mass GTP-binding proteins were detected by the radiolabeled GTP-blot overlay method of Bhullar and Haslam (1987), with modification as described by Schneffel et al (1992) and Gromov and Celis (1994). Briefly, SGM, PM, CY, CO, ER, rR, and molecular weight standards were separated by SDS-12% PAGE and transferred to nitrocellulose filters.…”

Section: Preparation Of Cytosolmentioning

confidence: 99%

Immunolocalization of Rap1 in the Rat Parotid Gland: Detection on Secretory Granule Membranes

D’Silva

DiJulio

Belton

et al. 1997

J Histochem Cytochem.

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WashingtonS U M M A R Y The objective of this study was to localize rap1 in the rat parotid gland. Rap1 is a small GTP-binding protein that has been linked to phagocytosis in neutrophils and various functions in platelets. In this study, we used [ ␣-32 P]-GTP-blot overlay analysis, immunoblot analysis, and immunohistochemistry to identify rap1 in rat parotid gland. The immunohistochemical techniques included immunoperoxidase and widefield microscopy with image deconvolution. Rap1 was identified in the secretory granule membrane (SGM), plasma membrane (PM), and cytosolic (CY) fractions, with the largest signal being in the SGM fraction. The tightly bound vs loosely adherent nature of SGM-associated rap1 was determined using sodium carbonate, and its orientation on whole granules was assessed by trypsin digestion. Rap1 was found to be a tightly bound protein rather than a loosely adherent contaminant protein of the SGM. Its orientation on the cytosolic face of the secretory granule (SG) is of significance in postulating a function for rap1 because exocytosis involves the fusion of the cytoplasmic face of the SG with the cytoplasmic face of the PM, with subsequent release of granule contents (CO). Therefore, the localization and high concentration of rap1 on the SGM and its cytosolic orientation suggest that it may play a role in the regulation of secretion.

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Detection of 23-27 kDa GTP-binding proteins in platelets and other cells

Cited by 167 publications

References 35 publications

Interaction of small G proteins with photoexcited rhodopsin

Interaction of small G proteins with photoexcited rhodopsin

GTP‐blot Analysis of Small GTP‐binding Proteins

Immunolocalization of Rap1 in the Rat Parotid Gland: Detection on Secretory Granule Membranes

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