Epinephrine induces changes in the subcellular distribution of the inhibitory GTP-binding protein Gi alpha-2 and a 38-kDa phosphorylated protein in the human platelet.
By using antibodies specific for alpha subunits of inhibitory GTP-binding proteins (Gi alpha polypeptides) to probe Western blots of whole platelet protein, we detected Gi alpha-2 as the predominant Gi alpha species present in platelets. The subcellular compartmentalization of distinct Gi alpha-2-immunoreactive polypeptides coupled to thrombin and alpha 2-adrenergic receptors was examined in Triton X-100 platelet lysates prepared by highspeed centrifugation. This treatment permitted separation of the Triton-insoluble membrane skeleton from Triton-soluble cell components. In cells treated with either alpha-thrombin or epinephrine, we observed that a greater proportion of Gi alpha-2 was localized in the Triton-soluble fraction than in the Triton-insoluble fraction. Pertussis toxin was found to catalyze ADP-ribosylation of Gi alpha-2 in whole platelets. In thrombin-stimulated cells, this activity was confined to the Triton-soluble fraction and was markedly lower than that of unstimulated cells. Epinephrine, on the other hand, promoted translocation of a portion of the pertussis toxin-sensitive Gi alpha-2 from the Triton-soluble fraction to the Triton-insoluble fraction. In addition, epinephrine stimulated translocation of a phosphorylated protein of approximately 38 kDa that was not ADP-ribosylated by pertussis toxin. This protein expressed immunoreactivity with the general Gi alpha antiserum AS/7 but not with the Gi alpha-2 antiserum LE/3. These findings suggest a role for specific localization of Gi alpha proteins in epinephrine-induced platelet responses.
Crouch
M F
MF
Division of Cell Biology, Burroughs Wellcome Co., Research Triangle Park, NC 27709.
Winegar
D A
DA
Lapetina
E G
EG
D000246:Adenosine Diphosphate Ribose / Q000097:blood; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004837:Epinephrine / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000097:blood*; D006801:Humans; D017830:Octoxynol; D037342:Pertussis Toxin; D010750:Phosphoproteins / Q000097:blood*; D010766:Phosphorylation; D011092:Polyethylene Glycols; D011942:Receptors, Adrenergic, alpha / Q000378:metabolism; D012995:Solubility; D013347:Subcellular Fractions / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D010750:Phosphoproteins; D011942:Receptors, Adrenergic, alpha; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D011092:Polyethylene Glycols; D017830:Octoxynol; D037342:Pertussis Toxin; D013917:Thrombin; D019204:GTP-Binding Proteins; D004837:Epinephrine