Farnesylcysteine analogues inhibit store-regulated Ca2+ entry in human platelets: evidence for involvement of small GTP-binding proteins and actin cytoskeleton.
We have investigated the mechanism of Ca(2+) entry into fura-2-loaded human platelets by preventing the prenylation of proteins such as small GTP-binding proteins. The farnesylcysteine analogues farnesylthioacetic acid (FTA) and N-acetyl-S-geranylgeranyl-L-cysteine (AGGC), which are inhibitors of the methylation of prenylated and geranylgeranylated proteins respectively, significantly decreased thrombin-evoked increases in intracellular free Ca(2+) concentration ([Ca(2+)](i)) in the presence, but not in the absence, of external Ca(2+), suggesting a relatively selective inhibition of Ca(2+) entry over internal release. Both these compounds and N-acetyl-S-farnesyl-L-cysteine, which had similar effects to those of FTA, also decreased Ca(2+) entry evoked by the depletion of intracellular Ca(2+) stores with thapsigargin. The inactive control N-acetyl-S-geranyl-L-cysteine was without effect. Patulin, an inhibitor of prenylation that is inert with respect to methyltransferases, also decreased store-regulated Ca(2+) entry. Cytochalasin D, an inhibitor of actin polymerization, significantly decreased store-regulated Ca(2+) entry in a time-dependent manner. Both cytochalasin D and the farnesylcysteine analogues FTA and AGGC inhibited actin polymerization; however, when evoking the same extent of decrease in actin filament formation, FTA and AGGC showed greater inhibitory effects on Ca(2+) entry, indicating a cytoskeleton-independent component in the regulation of Ca(2+) entry by small GTP-binding-protein. These findings suggest that prenylated proteins such as small GTP-binding proteins are involved in store-regulated Ca(2+) entry through actin cytoskeleton-dependent and cytoskeleton-independent mechanisms in human platelets.
Rosado
J A
JA
Department of Physiology, University of Cambridge, Downing Street, Cambridge CB2 3EG, U.K.
Sage
S O
SO
D000199:Actins / Q000097:blood* / Q000187:drug effects; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D002118:Calcium / Q000097:blood*; D003545:Cysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects / Q000502:physiology*; D004224:Diterpenes / Q000494:pharmacology*; D005204:Farnesol / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D020559:Monomeric GTP-Binding Proteins / Q000097:blood*; D019000:Phosphotyrosine / Q000097:blood; D011496:Protein Methyltransferases / Q000037:antagonists & inhibitors; D017368:Protein Prenylation / Q000187:drug effects; D019284:Thapsigargin / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology
D000199:Actins; D004224:Diterpenes; C095716:N-acetyl-S-geranylgeranyl-cysteine; C112353:farnesylthioacetic acid; D019000:Phosphotyrosine; D015638:Cytochalasin D; D005204:Farnesol; D019284:Thapsigargin; D011496:Protein Methyltransferases; D013917:Thrombin; D020559:Monomeric GTP-Binding Proteins; D003545:Cysteine; D002118:Calcium