RAP2B: a RAS-related GTP-binding protein from platelets.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 87 ·No. 17 ·1990-09-00 ·Pages 6527-31

Ohmstede CA, Farrell FX, Reep BR, Clemetson KJ, Lapetina EG

Abstract

A platelet cDNA expression library was screened with the monoclonal antibody M90, which recognizes a specific epitope on RAS-encoded p21 proteins (amino acids 107-130). DNA sequence analysis of one clone revealed that it encoded a partial amino acid sequence of a protein closely related to RAP2, which we have named RAP2B. A repeated screening of the platelet cDNA library with an internal Ava I fragment of the RAP2B cDNA allowed the isolation of a full-length cDNA for the RAP2B sequence. RAP2B is 90% identical to RAP2 at the amino acid level with the most variability at the carboxyl terminus of the protein. Oligonucleotides were synthesized to complete the amino acid sequence of the RAP2B protein and the entire sequence was expressed in Escherichia coli. Analysis of crude soluble extracts indicated that RAP2B was a Mr 22,000 protein that specifically bound GTP on blots. Moreover, incubation of similar extracts with the catalytic subunit of cAMP-dependent protein kinase did not cause phosphorylation of RAP2B, as had been observed for the closely homologous proteins, RAP1A and RAP1B. These results suggest that RAP2B, like the other RAP proteins, is a low molecular weight GTP-binding protein in human platelets.

MeSH Terms

Amino Acid Sequence Base Sequence Blood Platelets/metabolism Cloning, Molecular GTP-Binding Proteins/blood,genetics,isolation & purification Gene Library Genes, ras Humans Molecular Sequence Data Molecular Weight Oligonucleotide Probes Protein Kinases/metabolism Restriction Mapping Sequence Homology, Nucleic Acid rap GTP-Binding Proteins

Chemicals

Oligonucleotide Probes Protein Kinases GTP-Binding Proteins RAP2B protein, human rap GTP-Binding Proteins

Authors & Affiliations

5 authors, click to expand affiliations / ORCID

Ohmstede C A

Division of Cell Biology, Burroughs Wellcome Co., Research Triangle Park, NC 27709.

Farrell F X

Reep B R

Clemetson K J

Lapetina E G

References (28)

28 references, click to expand

A novel small molecular weight GTP-binding protein with the same putative effector domain as the ras proteins in bovine brain membranes. Purification, determination of primary structure, and characterization.
J Biol Chem. 1988 Dec 15;263(35):18965-71 PMID: 3143720
Isolation and characterization of human blood platelet mRNA and construction of a cDNA library in lambda gt11. Confirmation of the platelet derivation by identification of GPIb coding mRNA and cloning of a GPIb coding cDNA insert.
Thromb Haemost. 1989 Jun 30;61(3):448-53 PMID: 2799758
Phosphorylation by cyclic AMP-dependent protein kinase of a human platelet Mr 22,000 GTP-binding protein (smg p21) having the same putative effector domain as the ras gene products.
Biochem Biophys Res Commun. 1988 Dec 30;157(3):851-60 PMID: 2849942
A ras-related gene with transformation suppressor activity.
Cell. 1989 Jan 13;56(1):77-84 PMID: 2642744
Cloning, purification, and properties of Candida albicans thymidylate synthase.
J Bacteriol. 1989 Mar;171(3):1372-8 PMID: 2646281
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature. 1970 Aug 15;227(5259):680-5 PMID: 5432063
DNA sequencing with chain-terminating inhibitors.
Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463-7 PMID: 271968
Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350-4 PMID: 388439
Biological and biochemical properties of human rasH genes mutated at codon 61.
Cell. 1986 Jan 17;44(1):167-76 PMID: 3510078
Purification of the major GTP-binding proteins from human placental membranes.
J Biol Chem. 1986 May 25;261(15):7052-9 PMID: 3084494
ras p21 deletion mutants and monoclonal antibodies as tools for localization of regions relevant to p21 function.
Proc Natl Acad Sci U S A. 1986 Aug;83(15):5400-4 PMID: 2426696
Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes.
J Mol Biol. 1986 May 5;189(1):113-30 PMID: 3537305
Specific binding of [alpha-32P]GTP to cytosolic and membrane-bound proteins of human platelets correlates with the activation of phospholipase C.
Proc Natl Acad Sci U S A. 1987 Apr;84(8):2261-5 PMID: 3470789
ras genes.
Annu Rev Biochem. 1987;56:779-827 PMID: 3304147
Detection of 23-27 kDa GTP-binding proteins in platelets and other cells.
Biochem J. 1987 Jul 15;245(2):617-20 PMID: 3117049
At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells.
J Mol Biol. 1987 Aug 20;196(4):947-50 PMID: 3681984
Posttranslational modification of the Ha-ras oncogene protein: evidence for a third class of protein carboxyl methyltransferases.
Proc Natl Acad Sci U S A. 1988 Jul;85(13):4643-7 PMID: 3290900
Human cDNAs rap1 and rap2 homologous to the Drosophila gene Dras3 encode proteins closely related to ras in the 'effector' region.
Oncogene. 1988 Aug;3(2):201-4 PMID: 3045729
Nucleotide sequence of a human cDNA encoding a ras-related protein (rap1B).
Nucleic Acids Res. 1988 Aug 11;16(15):7719 PMID: 3137530
Gn-proteins are distinct from ras p21 and other known low molecular mass GTP-binding proteins in the platelet.
FEBS Lett. 1988 Sep 12;237(1-2):168-72 PMID: 3139450
Low Mr GTP-binding proteins in human platelets: cyclic AMP-dependent protein kinase phosphorylates m22KG(I) in membrane but not c21KG in cytosol.
Biochem Biophys Res Commun. 1989 Apr 14;160(1):235-42 PMID: 2540745
A ras-related protein is phosphorylated and translocated by agonists that increase cAMP levels in human platelets.
Proc Natl Acad Sci U S A. 1989 May;86(9):3131-4 PMID: 2470091
All ras proteins are polyisoprenylated but only some are palmitoylated.
Cell. 1989 Jun 30;57(7):1167-77 PMID: 2661017
Agonist-induced phosphorylation of an immunologically ras-related protein in human erythroleukemia cells.
Biochem Biophys Res Commun. 1989 Jun 30;161(3):972-8 PMID: 2472791
Post-translational processing of p21ras is two-step and involves carboxyl-methylation and carboxy-terminal proteolysis.
EMBO J. 1989 Apr;8(4):1093-8 PMID: 2663468
Genetic and pharmacological suppression of oncogenic mutations in ras genes of yeast and humans.
Science. 1989 Jul 28;245(4916):379-85 PMID: 2569235
Phosphorylation of smg p21, a ras p21-like GTP-binding protein, by cyclic AMP-dependent protein kinase in a cell-free system and in response to prostaglandin E1 in intact human platelets.
J Biol Chem. 1989 Sep 15;264(26):15688-95 PMID: 2504724
Identification of a platelet Mr 22,000 GTP-binding protein as the novel smg-21 gene product having the same putative effector domain as the ras gene products.
Biochem Biophys Res Commun. 1988 Dec 15;157(2):670-6 PMID: 3144274

Article Info

Journal

Proceedings of the National Academy of Sciences of the United States of America

Abbr.

Proc Natl Acad Sci U S A

ISSN

0027-8424

DOI

10.1073/pnas.87.17.6527

Published

1990-09-00

Pages

6527-31

Language

English

Region

United States

NLM ID

7505876

PMCID

PMC54569

Subset

Databases

GENBANK

X52987

External Links

PubMed source DOI: 10.1073/pnas.87.17.6527 PMC Full Text View journal details

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RAP2B: a RAS-related GTP-binding protein from platelets.

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