The interaction of fibrinogen with the integrin ␣ IIb ␤ 3 plays a crucial role in platelet adhesion and platelet activation leading to the generation of intracellular signals that nucleate the reorganization of the cytoskeleton. Presently, we have only a limited understanding of the signaling cascades and effector proteins through which changes in the cytoskeletal architecture are mediated. The present study identifies phospholipase C␥2 (PLC␥2) as an important target of the Src-dependent signaling cascade regulated by ␣ IIb ␤ 3 . Real time phasecontrast microscopy is used to show that formation of filopodia and lamellapodia in murine platelets on a fibrinogen surface is dramatically inhibited in the absence of PLC␥2. Significantly, the formation of these structures is mediated by Ca 2؉ elevation and activation of protein kinase C, both directly regulated by PLC activity. With the involvement of Syk, SLP-76, and Btk, ␣ IIb ␤ 3 -induced PLC␥2 activation partly overlaps with the pathway used by the collagen receptor glycoprotein VI. Important differences, however, exist between the two signaling cascades in that activation of PLC␥2 by ␣ IIb ␤ 3 is unaltered in murine platelets, which lack the FcR ␥-chain or the adaptor LAT, but is abolished in the presence of cytochalasin D. Therefore, PLC␥2 plays not only a crucial role in activation of ␣ IIb ␤ 3 by collagen receptors but also in ␣ IIb ␤ 3 -mediated responses.The integrin ␣ IIb ␤ 3 mediates platelet aggregation in cell suspensions and supports adhesion to fibrinogen and von Willebrand factor (vWf) 1 -coated surfaces. On resting platelets, the integrin is in a low affinity conformation that does not support binding to either adhesion molecule at their normal plasma concentrations. An increase in affinity of ␣ IIb ␤ 3 , leading to fibrinogen and vWf binding, is mediated by inside-out signals from G protein-coupled and tyrosine kinase-linked agonists, and thereby promotes aggregation. In addition, binding to ␣ IIb ␤ 3 induces outside-in signals that lead to reorganization of the cytoskeleton and synergize with other agonists to mediate activation. The central role of the integrin ␣ IIb ␤ 3 in thrombosis and hemostasis is highlighted by the severe bleeding disorders in patients with Glanzmann thrombasthenia, which lack functional integrin.One of the earlier events to occur following ligation of ␣ IIb ␤ 3 is the activation of the tyrosine kinase Syk via one or more Src kinases (1, 2). This leads to tyrosine phosphorylation of the adaptor molecule SLP-76, which is constitutively associated with a second adaptor SLAP-130 (3, 4), also known as Fynbinding protein or adhesion-and degranulation-promoting adapter protein (5, 6). Together with proteins of the Vav GTPase exchange family, the adapter Nck, and the actin-binding protein VASP, this cascade has been shown to lead to activation of phosphoinositol 3-kinase and phosphorylation of FAK, and subsequent reorganization of the cytoskeleton in ␣ IIb ␤ 3 -transfected Chinese hamster ovary cells (7-9). Evidence that this casc...

The platelet collagen receptor glycoprotein VI (GPVI) and the fibrinogen receptor integrin alphaIIbbeta3 trigger intracellular signalling cascades involving the tyrosine kinase Syk, the adapter SLP-76 and phospholipase Cgamma2 (PLCgamma2). Similar pathways are activated downstream of immune receptors in lymphocytes, where they have been localized in part to glycolipid-enriched membrane domains (GEMs). Here we provide several lines of evidence that GPVI-mediated tyrosine phosphorylation of PLCgamma2 in platelets is dependent on GEM-organized signalling and utilizes the GEM resident adapter protein LAT (linker for activation of T cells). In sharp contrast, although fibrinogen binding to platelets stimulates alphaIIbbeta3-dependent activation of Syk and tyrosine phosphorylation of SLP-76 and PLCgamma2, it does not utilize GEMs to promote these responses or to support platelet aggregation. These results establish that GPVI and alphaIIbbeta3 trigger distinct patterns of receptor signalling in platelets, leading to tyrosine phosphorylation of PLCgamma2, and they highlight the role of GEMs in compartmentalizing signalling reactions involved in haemostasis.

The actual frequency of constitutively activating thyrotropin receptor or Gsalpha mutations in toxic thyroid nodules (TTNs) remains controversial as considerable variation in the prevalence of these mutations has been reported. We studied a series of 75 consecutive TTNs and performed mutation screening by the more sensitive method of denaturing gradient gel electrophoresis (DGGE) in addition to direct sequencing. Furthermore, the likelihood of somatic mutations occurring in genes other than that for the thyroid-stimulating hormone receptor (TSHR) and exons 7-9 of the Gsalpha protein gene was determined by clonality analysis of TTNs, which did not harbor mutations in the investigated genes. In 43 of 75 TTNs (57%) constitutively active TSHR mutations were identified. Six TSHR mutations were detected only by DGGE, underlining the importance of a sensitive screening method. Novel, constitutively activating mutations were identified at positions 425 (Ser-->Leu) and 512 (Leu-->Glu/Arg). Furthermore, a new base substitution was detected at position Pro639Ala (CCA-->GCA). Ten of 20 TSHR or Gsalpha mutation negative cases (50%) showed nonrandom X-chromosome inactivation, indicating clonal origin. In conclusion, somatic, constitutively activating TSHR mutations appear to be a major cause of TTNs (57%), while mutations in Gsalpha play a minor role (3%). The mutation negative but clonal cases indicate a probable involvement of somatic mutations other than in the TSH receptor or Gsalpha genes as the molecular cause of these hot nodules.

Gain-of-function mutations of the thyrotropin receptor (TSHR) gene have been invoked as one of the major causes of toxic thyroid adenomas. In a toxic thyroid nodule, we recently identified a 9-amino acid deletion (amino acid positions 613-621) within the third intracellular (i3) loop of the TSHR resulting in constitutive receptor activity. This finding exemplifies a new mechanism of TSHR activation and raises new questions concerning the function of the i3 loop. Because the i3 loop is thought to be critical for receptor/G protein interaction in many receptors, we systematically reexamined the role of the TSHR's i3 loop for G protein coupling. Thus, various deletion mutants were generated and functionally characterized. We identified an optimal deletion length responsible for constitutive activity. If the number of deleted amino acids was reduced, elevated basal cAMP accumulation was found to be concomitantly diminished. Expansion of the deletion dramatically impaired cell surface expression of the receptor. Shifting the deletion toward the N terminus of the i3 loop resulted in unaltered strong constitutive receptor activity. In contrast, translocation of the deletion toward the C terminus led to significantly reduced basal cAMP formation, most probably due to destruction of a conserved cluster of amino acids. In this study, we show for the first time that amino acid deletions within the i3 loop of a G protein-coupled receptor result in constitutive receptor activity. In the TSHR, 75% of the i3 loop generally assumed to play an essential role in G protein coupling can be deleted without rendering the mutant receptor unresponsive to thyrotropin. These findings support a novel model explaining the molecular events accompanying receptor activation by agonist.

Neonatal hyperthyroidism in the absence of maternal autoimmune thyroid disease and without thyroid-stimulating antibodies in the child is rare. We here describe a boy with severe intrauterine hyperthyroidism and advanced bone age in the absence of thyroid-stimulating autoantibodies. After long term antithyroid treatment and relapse of hyperthyroidism, a near-total thyroid resection was performed. The necessity to progressively decrease postoperative thyroid hormone replacement indicates thyroid tissue regrowth in the small thyroid remnant. Analysis of the genomic DNA of the child's peripheral leukocytes showed a G to A base exchange that led to a heterozygous Ser to Asn conversion at position 505 in the third transmembrane region of the TSH receptor (TSHR). The absence of the Ser505 Asn mutation in all other family members identifies the child's TSHR mutation as a sporadic germline mutation. Transient expression of the mutated TSH receptor in COS-7 cells showed a constitutively activated cAMP cascade. We thus identified a new constitutively activating germline mutation. Neonates with persistent nonautoimmune hyperthyroidism should be investigated for TSHR germline mutations. Because of frequent relapses, patients with sporadic congenital nonautoimmune hyperthyroidism should be treated with early subtotal to near-total thyroid resection. Moreover, post-operative radioiodine treatment should be considered.