Thrombolamban, the 22-kDa platelet substrate of cyclic AMP-dependent protein kinase, is immunologically homologous with the Ras family of GTP-binding proteins.
Platelet inhibition by agents that increase intracellular levels of cAMP is associated with cAMP-dependent phosphorylation of specific intracellular proteins, including a membrane-associated 22-kDa microsomal protein called thrombolamban. In view of recent studies suggesting that platelets also contain 22-kDa GTP-binding proteins that are homologous with ras-encoded p21 proteins, the present work was undertaken to examine the possibility that thrombolamban and the Ras-like proteins were the same. Platelet microsomes were labeled with [gamma-32P]ATP and the labeled proteins were examined by autoradiography of sodium dodecyl sulfate/polyacrylamide gels. On Western blots of both one-dimensional and two-dimensional gels, thrombolamban immunoreacted with M90, a monoclonal antibody that recognizes the GTP-binding domain of Ras p21 proteins, but not with Y13-259, a monoclonal antibody that recognizes another domain and is specific for Ras proteins. Overlay experiments with unlabeled platelet microsomes demonstrated numerous low molecular weight proteins that bound [alpha-32P]GTP, although none could be identified as thrombolamban. Finally, thrombolamban was immunoprecipitated by M90. These studies show that thrombolamban is a low molecular weight protein that is immunologically related to the Ras family of GTP-binding proteins.
Center for Thrombosis and Hemostasis, Department of Medicine, Dental Research Center, University of North Carolina, Chapel Hill 27599.
T E
TE
White
J C
JC
Lacal
B
B
Reep
T H
TH
Fischer
E G
EG
Lapetina
G C
GC
White
D000255:Adenosine Triphosphate / Q000097:blood; D000911:Antibodies, Monoclonal; D001792:Blood Platelets / Q000378:metabolism; D001798:Blood Proteins / Q000276:immunology / Q000378:metabolism*; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D000939:Epitopes / Q000032:analysis*; D019204:GTP-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007525:Isoelectric Focusing; D008861:Microsomes / Q000378:metabolism; D008970:Molecular Weight; D015689:Oncogene Protein p21(ras) / Q000276:immunology / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D001798:Blood Proteins; D002135:Calcium-Binding Proteins; D000939:Epitopes; C054412:thrombolamban; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D019204:GTP-Binding Proteins; D015689:Oncogene Protein p21(ras)