Structural and functional studies on the G(o) protein.
Monoclonal antibodies were raised against purified bovine and human brain G-proteins. The epitopes recognized by three monoclonal antibodies (MONO, 3C2, and 3E7) were mapped by expressing defined parts of the bovine G(o) alpha-cDNA in bacteria, followed by immunoblotting. All three antibodies recognize the recombinant bovine alpha o-protein, but at distinct sites. The epitopes of MONO and 3C2 were mapped between alpha o amino acids 80 and 145, and both antibodies recognize alpha o exclusively. Heterotrimeric G(o)-proteins as well as guanosine 5'-3-O-(thio)triphosphate-liganded free alpha o-subunits are readily immunoprecipitated by these monoclonal antibodies. Binding of MONO or 3C2 does not affect ADP-ribosylation of the alpha o-subunit by pertussis toxin. Apparently, the antibodies do not bind to or induce large conformational changes in regions of the alpha o-subunit that are involved in association with beta gamma-subunits or ADP-ribosylation. 3E7 behaves as an anti common alpha-subunit antibody when used in immunoblots. However, under nondenaturing conditions, 3E7 recognizes alpha o exclusively. After binding of 3E7, the pertussis toxin-dependent ADP-ribosylation of alpha o is effectively blocked, while the ADP-ribosylation of the various alpha i-subunits is not affected. The epitope of 3E7 was mapped between alpha o amino acids 13-88, a region which has been implicated in the interaction between alpha- and beta gamma-subunits. Possibly, the inhibitory effect of 3E7 on ADP-ribosylation of G(o) is secondary to the loss of beta gamma-subunits that is observed upon binding of 3E7.
van der Voorn
L
L
Division of Cellular Biochemistry, The Netherlands Cancer Institute, Amsterdam.
Tol
O
O
Hengeveld
T M
TM
Ploegh
H L
HL
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology; D000918:Antibody Specificity; D001921:Brain / Q000378:metabolism; D002417:Cattle; D000939:Epitopes / Q000276:immunology; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D037342:Pertussis Toxin; D011233:Precipitin Tests; D017386:Sequence Homology, Amino Acid; D010566:Virulence Factors, Bordetella / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins