Platelets are small disc-shaped cell fragments which undergo a rapid transformation when they encounter vascular damage. They become more spherical and extrude pseudopodia, their fibrinogen receptors are activated, causing them to aggregate, they release their granule contents, and eventually form a plug which is responsible for primary haemostasis. Activation of platelets is also implicated in the pathogenesis of unstable angina, myocardial infarction and stroke. Here we show that platelets from mice deficient in the alpha-subunit of the heterotrimeric guanine-nucleotide-binding protein Gq are unresponsive to a variety of physiological platelet activators. As a result, G alpha(q)-deficient mice have increased bleeding times and are protected from collagen and adrenaline-induced thromboembolism. We conclude that G alpha(q) is essential for the signalling processes used by different platelet activators and that it cannot be replaced by G alpha(i) or the beta gamma subunits of the heterotrimeric G proteins. G alpha(q) may thus be a new target for drugs designed to block the activation of platelets.

The ubiquitously expressed G-proteins G12 and G13 whose function is currently not clear have been shown to be activated in platelet membranes through receptors that stimulate platelet aggregation. We used intact human platelets to determine whether alpha subunits of both G-proteins can be phosphorylated under physiological conditions. Activation of human platelets by thrombin and the thromboxane A2 receptor agonist U46619 lead to phosphorylation of Galpha12 and Galpha13. Phosphorylation occurred rapidly after addition of thrombin and was not mediated by glycoprotein IIb/IIIa (integrin alphaIIbbeta3) activation. Phosphorylation of Galpha12 and Galpha13 could be mimicked by phorbol 12-myristate 13-acetate, and thrombin-induced phosphorylation was inhibited by the protein kinase C inhibitor calphostin C indicating an involvement of protein kinase C in Galpha12/13 phosphorylation induced by thrombin in human platelets. The phosphorylation of both G protein alpha subunits was reconstituted in COS-7 cells cotransfected with Galpha12 or Galpha13 and different protein kinase C isoforms. Among the protein knase C isoforms tested, protein kinase C beta, delta, and epsilon were most effective in promoting phosphorylation of Galpha12 and Galpha13 in a phorbol 12-myristate 13-acetate-dependent manner. These data demonstrate that Galpha12 and Galpha13 are phosphorylated under in vivo conditions and that this phosphorylation involves protein kinase C.

The conformation and activity of pRb, the product of the retinoblastoma susceptibility gene, is dependent on the phosphorylation status of one or more of its 16 potential cyclin-dependent kinase (cdk) sites. However, it is not clear whether the phosphorylation status of one or more of these sites contributes to the determination of the various conformations and activity of pRb. Moreover, whether and how the conformation of pRb may regulate the phosphorylation of the cdk sites is also unclear. In the process of analyzing the function and regulation of pRb, we uncovered the existence of an unusual structural motif, m89 (amino acids 880 -900), the mutation of which confers upon pRb a hypophosphorylated conformation. Mutation of this structural domain activates, rather than inactivates, the growth suppressor function of pRb. In order to understand the effect of the mutation of m89 on the phosphorylation of cdk sites, we identified all the cdk sites (Thr-356, Ser-807/Ser-811, and Thr821) the phosphorylation of which drastically modify the conformation of pRb. Mutation of each of these four sites alone or in combinations results in the different conformations of pRb, the migration pattern of which, on SDS-polyacrylamide gel electrophoresis, resembles various in vivo hypophosphorylated forms. Each of these hypophosphorylated forms of pRb has enhanced growth suppressing activity relative to the wild type. Our data revealed that the m89 structural motif controls the exposure of the cdk sites Ser-807/Ser-811 in vitro and in vivo. Moreover, the m89 mutant has enhanced growth suppressing activity, similar to a mutant with alanine substitutions at Ser-807/Ser-811. Our recent finding, that the m89 region is part of a structural domain, p5, conserved antigenically and functionally between pRb and p53, suggests that the evolutionarily conserved p5 domain may play a role in the coordinated regulation of the activity of these two tumor suppressors, under certain growth conditions.

The mammalian G proteins G15 and G16 couple a wide variety of receptors to phospholipase C (PLC) in co-transfected systems, and it has been suggested that they can be used as tools in agonist-screening systems. Using the reversed tetracycline-controlled transactivation system we generated rat pituitary GH3 cell clones that expressed Galphal5 and Galpha16 conditionally to study the coupling of endogenous receptors to both G proteins. In cells expressing moderate levels of Galpha15, activation of various endogenous receptors increased inositol phosphate production, whereas conditional expression of Galpha16 had no significant effect on agonist-dependent PLC activity. Activation of PLC through Galpha15 in response to carbachol did not increase cytosolic [Ca2+] ([Ca2+]i) but stimulated protein kinase C. While carbachol decreased the secretory activity in non-induced GH3 cells, it increased secretion in cells expressing Galpha15. Our data demonstrate that Galpha15 has a higher functional promiscuity than Galpha16 when studied in a system that preserves physiological G protein and receptor levels. In addition, Galpha15-mediated coupling of a receptor to PLC can change the cellular response to receptor agonists, indicating that downstream cellular functions can be used to detect receptor activation in screening systems employing a promiscuous G protein.