title: Interactions in platelets between G proteins and the agonists that stimulate phospholipase C and inhibit adenylyl cyclase.
abstract: Platelet responses to agonists are believed to be mediated by at least two pertussis toxin-sensitive guanine nucleotide-binding (G) proteins: Gi which inhibits adenylyl cyclase and Gp, which stimulates phospholipase C. The present studies compare the properties of Gi and Gp and examine their interactions with the receptors for various platelet agonists. In permeabilized platelets and platelet membranes, pertussis toxin [32P]ADP-ribosylated a protein(s) (alpha 41) which migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis fractionally below rabbit and bovine alpha i (Mr = 41,000). Prior exposure of the platelets to an agonist inhibited the [32P]ADP-ribosylation of alpha 41 to an extent which correlated with the pattern of responses to that agonist. Thrombin, which elicited responses that were mediated by both Gi and Gp, decreased radiolabeling by greater than 90%. Epinephrine, which was functionally coupled only to Gi, decreased radiolabeling by 50%, as did vasopressin and platelet-activating factor (PAF), which were coupled only to Gp. U46619, a thromboxane analog which neither inhibited cAMP formation nor caused pertussis toxin-sensitive phosphoinositide hydrolysis, had no effect on 32P-ADP-ribosylation. These results suggest that either G alpha 41 regulates more than one enzyme or that alpha subunits from more than one G protein comigrate within alpha 41. Two-dimensional electrophoresis was used to test the latter possibility. Upon isoelectric focusing, alpha 41 resolved into two distinct subspecies. However, these appear to be minor variants rather than functionally distinct alpha subunits since: 1) both proteins produced the same proteolytic fragments after digestion with chymotrypsin or Staphylococcus aureus V8 protease and 2) preincubation of the platelets with agonists, including those which appear to interact in intact platelets solely with Gp (PAF and vasopressin) or solely with Gi (epinephrine), inhibited the [32P]ADP-ribosylation of both proteins to the same extent. The pattern of functional responses produced by some of the agonists was found to depend upon the conditions used for the assay. Although unable to inhibit cAMP formation in intact platelets, both PAF and vasopressin caused pertussis toxin-sensitive inhibition of adenylyl cyclase in isolated membranes. Collectively, these observations suggest that 1) in platelets a single pertussis toxin-sensitive, alpha 41-containing G protein may be involved in the regulation of both adenylyl cyclase and phospholipase C and 2) additional constraints which are altered during membrane isolation may help to determine which enzyme is coupled to which agonist.
authors.affiliation: Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104-4283.
authors.forename: L F
authors.initials: LF
authors.lastname: Brass
authors.forename: M J
authors.initials: MJ
authors.lastname: Woolkalis
authors.forename: D R
authors.initials: DR
authors.lastname: Manning
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000097:blood; D001792:Blood Platelets / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D000242:Cyclic AMP / Q000097:blood; D011464:Epoprostenol / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000097:blood*; D006801:Humans; D010449:Peptide Mapping; D037342:Pertussis Toxin; D010972:Platelet Activating Factor / Q000494:pharmacology; D010974:Platelet Aggregation; D011450:Prostaglandin Endoperoxides, Synthetic / Q000494:pharmacology; D010738:Type C Phospholipases / Q000097:blood*; D014667:Vasopressins / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D010972:Platelet Activating Factor; D011450:Prostaglandin Endoperoxides, Synthetic; D010566:Virulence Factors, Bordetella; D014667:Vasopressins; D000246:Adenosine Diphosphate Ribose; D011464:Epoprostenol; D000242:Cyclic AMP; D037342:Pertussis Toxin; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases