We have generated mouse transgenic lineages for C3G (tgC3G) and C3GΔCat (tgC3GΔCat, C3G mutant lacking the GEF domain), where the transgenes are expressed under the control of the megakaryocyte and platelet specific PF4 (platelet factor 4) gene promoter. Transgenic platelet activity has been analyzed through in vivo and in vitro approaches, including bleeding time, aggregation assays and flow cytometry. Both transgenes are expressed (RNA and protein) in purified platelets and megakaryocytes and do not modify the number of platelets in peripheral blood. Transgenic C3G animals showed bleeding times significantly shorter than control animals, while tgC3GΔCat mice presented a remarkable bleeding diathesis as compared to their control siblings. Accordingly, platelets from tgC3G mice showed stronger activation in response to platelet agonists such as thrombin, PMA, ADP or collagen than control platelets, while those from tgC3GΔCat animals had a lower response. In addition, we present data indicating that C3G is a mediator in the PKC pathway leading to Rap1 activation. Remarkably, a significant percentage of tgC3G mice presented a higher level of neutrophils than their control siblings. These results indicate that C3G plays an important role in platelet clotting through a mechanism involving its GEF activity and suggest that it might be also involved in neutrophil development.

The platelet fibrinogen receptor, integrin ␣ IIb ␤ 3 , is a noncovalent heterodimer of glycoproteins IIb and IIIa. This work was aimed at elucidating the role played by the carboxy-terminal extracellular, transmembrane, and cytoplasmic regions of the glycoprotein ␤ 3 in the formation of functional complexes with ␣ subunits. Progressive carboxy-terminal deletions of ␤ 3 revealed that surface exposure of ␣ IIb ␤ 3 or ␣ v ␤ 3 could not occur in the absence of the transmembrane domain of ␤ 3 . In con- IntroductionThe glycoprotein (GP) IIb-IIIa complex, integrin ␣ IIb ␤ 3 , is a calcium-dependent, noncovalent heterodimer formed by GPIIb and GPIIIa. This complex is found in the plasma membrane of megakaryocytes, platelets, and some tumor tissues 1-3 and functions as a receptor for fibrinogen and other adhesive proteins like the von Willebrand factor, fibronectin, or vitronectin. 4 The ␤ 3 subunit may also complex the GP ␣ v to form the vitronectin receptor (integrin ␣ v ␤ 3 ) that shares with ␣ IIb ␤ 3 the binding of fibrinogen although with different affinity. 5 The platelet ␣ IIb ␤ 3 complex is essential to maintain a normal hemostasis. Unlike other platelet receptors that are constitutively active, the ␣ IIb ␤ 3 is maintained in a low-affinity state for its ligands. Disruption of the vascular endothelium and exposure of platelets to the action of agonists and adhesive proteins from the subendothelial matrix induces a cellular activation. The activated cells interact with adhesive proteins from the extracellular matrix, 6,7 and the ␣ IIb ␤ 3 receptors are able to bind fibrinogen with high affinity (insideout signaling), resulting in platelet aggregation. 8 Conversely, ligand-bound ␣ IIb ␤ 3 propagates signals to the interior of the cell (outside-in signaling) leading to enhanced interaction with the cytoskeleton, clustering of receptors (increased ligand avidity), and formation of focal contacts rich in signaling complexes. 9,10 The agonist-induced increase in ligand affinity of ␣ IIb ␤ 3 is thought to be the result of conformational changes of the heterodimer [11][12][13] initiated by the interaction of the cytoplasmic tails of ␣ and ␤ 3 subunits with cytosolic proteins. Despite the pathophysiologic importance of the platelet ␣ IIb ␤ 3 receptor, the knowledge of the mechanisms controlling its state of activation is rather limited.Unlike previous reports, 14 recent work from our laboratory 15 showed that a truncated form of ␤ 3 lacking the transmembrane and cytosolic domains failed to associate with ␣ IIb . The present work was aimed at further investigating the role played by the carboxyterminal domain of ␤ 3 in the surface expression and function of ␤ 3 heterodimers. The results obtained in this study indicate that surface expression of ␣ IIb ␤ 3 could not occur in the absence of the transmembrane domain of ␤ 3 . The present study has also revealed that either deletion of the carboxy-terminal region of the ␤ 3 ectodomain or disruption of the 663-687 disulfide bridge confers constitutive activity to the ␤ 3 integr...

Activin-A, a homodimeric protein composed of two inhibin beta A-subunits, was first isolated from gonadal fluids based upon its ability to stimulate FSH secretion and biosynthesis, but was also observed to suppress GH secretion. The present report describes the effects of activin on the biosynthesis of GH and the proliferation of pituitary somatotrophs. In pituitary cells cultured in the presence of 0.7 nM activin for 3 days, GH secretion was decreased by 50% compared to the control value. Inhibition of GH biosynthesis, measured by quantitative immunoprecipitation of [35S]methionine-labeled cells, could be observed after 24 h of activin treatment, and maximal (70%) inhibition of GH biosynthesis was observed after 3 days. Activin inhibited basal as well as GH-releasing factor (GRF)-, glucocorticoid-, and thyroid hormone-stimulated GH biosynthesis. Inhibin, which is known to reverse the effect of activin on FSH secretion, did not reverse the effect of activin on GH biosynthesis. Treatment of somatotrophs with activin for 3 days completely inhibited the growth-promoting effect of GRF on somatotrophs. However, no effect of activin on GRF-stimulated expression of the c-fos protooncogene was observed. These data demonstrate that activin, in addition to its stimulatory effect on FSH secretion, is able to inhibit both expression of GH and growth of somatotropic cells.

No abstract