No abstract

Bernard-Soulier Syndrome (BSS) is a rare congenital bleeding disorder due to absent or decreased expression of the glycoprotein Ib-IX-V (GpIb-IX-V) receptor complex on the platelet surface. To date, only mutations in GpIbalpha or GpIX have been reported in patients with BSS. GpIbbeta differs from the other proteins in this receptor in that the gene is more complex, and an alternative form is expressed in cells of non-megakaryocytic lineage, including endothelial cells. It appears that the megakaryocytic and endothelial cell mRNA species are transcribed from different start sites and have different proximal promoter regions. We have identified a patient with BSS who has a deletion on one chromosome 22, resulting in velocardiofacial syndrome. The GpIbbeta gene has been mapped to this deleted (22q11.2) region of chromosome 22. The patient has greatly reduced levels of GpIbbeta mRNA and no detectable platelet GpIbbeta protein, suggesting that his BSS results from a mutation in his remaining GpIbbeta allele. Sequence analysis revealed that the coding region of GpIbbeta is normal, but the 5'-upstream region contains a C to G transversion at base -133 from the transcription start site used in megakaryocytes. The mutation changes a GATA consensus binding site, disrupts GATA-1 binding to the mutated site, and decreases promoter activity by 84%. Thus, in this patient, Bernard-Soulier syndrome results from a deletion of one copy of GpIbbeta and a mutated GATA binding site in the promoter of the remaining allele, resulting in decreased promoter function and GpIbbeta gene transcription.

Mutation of the autophosphorylation sites of receptor protein-tyrosine kinases alters ligand dependent internalization and down-regulation, indicating a critical role for these sites in receptor processing. Currently, no differences in receptor processing based on an individual autophosphorylation site have been defined. By using a glutathione S-transferase fusion protein containing the src homology 2 domains of phospholipase C-␥ 1 to specifically recognize tyrosine 992 on the EGF receptor (Tyr(P) 992 ), we have found differences in this subpopulation of receptors. Following EGF stimulation, the number of Tyr(P) 992 receptors increased 2-fold over receptors identified by an antibody that recognizes activated EGF receptors (␣-Act. EGFR) in A431 cells. Confocal fluorescence microscopy showed that Tyr(P) 992 receptors underwent endocytosis at a slower rate and did not rapidly concentrate in juxtanuclear bodies. Tyr(P) 992 receptors were associated with more SOS, RasGTPase activating protein, phosphatidylinositol 3-kinase, and SHPTP2/syp, but less Grb2, than receptors in the general population, and these receptors were more heavily phosphorylated than the general population of active receptors. These findings suggest that autophosphorylation status is relevant to the endocytosis, degradation, and effector molecule interaction of individual EGF receptors. Further investigations based on phosphorylation status should provide new insights into how receptor protein-tyrosine kinase signaling is regulated. Receptor protein-tyrosine kinases (RPTKs)1 play an essential role in normal cell growth and neoplasia (1). Following ligand activation, these receptors oligomerize, activate, autophosphorylate, and rapidly endocytose (1-6). Autophosphorylation sites play an important role in signaling as well, since they serve as binding sites for proteins that contain src homology-2 (SH2) domains (7), which in turn propagate the signals of the receptor. SH2 domains are found in many different proteins including enzymes, transcription factors, and adaptor proteins (8, 9). At least eight SH2 proteins bind to the epidermal growth factor (EGF) receptor following activation. Each SH2 domain exhibits specificity based on amino acids surrounding the anchor phosphotyrosine, so the phosphorylation status of the receptor dictates subsequent interactions (10, 11). Interestingly, site-directed mutagenesis on the EGF receptors' autophosphorylation sites has shown that this receptor can be processed differently when these sites are altered. Mutation of Tyr 1068 , Tyr 1148 , or Tyr 1173 slightly prolongs the half-life of this receptor (12, 13), and deletion of all three results in a significant increase in half-life.Several studies have now shown that receptor internalization plays a role in signaling. Upon activation, receptors are endocytosed in clathrin-coated pits, transferred to endosomes, and then multivesicular bodies, which ultimately fuse with lysosomes (5,14,15). Because the autophosphorylation sites and kinase domain of the receptor are...

Exposure to shear stress has been shown to alter the expression of a number of surface components of cultured endothelial cells (EC). However, relatively few studies have examined the status of human EC surface proteins after prolonged flow, more closely corresponding to the steady state in vivo. Since the promoter region of glycoprotein (Gp) Ib alpha contains several copies of a putative shear stress response element, 5'-GAGACC-3', we investigated the response of cultured human umbilical vein EC (HUVEC) GpIb alpha to shear stress over a 72 h time period. In response to 30 dynes/cm2 of shear stress, total cell content of GpIb alpha protein was markedly increased above static levels at 7 and 24 h, as determined immunohistochemically. Western blot analysis of whole cell lysates after 24, 48, and 72 h of shear treatment demonstrated a 2.4-, 4.1-, and 3.2-fold increase in total GpIb alpha protein, respectively. Cell surface protein expression of GpIb alpha increased 2.5-fold at 7 h, as measured by quantitative immunofluorescence, and remained at that level at 24 h. After 48 h of shear stress, cell surface GpIb alpha, GpIX, and GpV, analyzed by flow cytometric analysis, were further increased over the levels observed at 24 h. The increase in cell surface membrane expression of GPIb alpha at 24, 48, and 72 h was confirmed by immunoprecipitation of biotinylated surface proteins. No upregulation of GpIb alpha was noted after exposure to shear stress of 1-3 dynes/cm2. These observations imply that under steady-state arterial shear conditions endothelial expression of the GpIb complex is significantly greater than observed in static EC cultures, and raise the possibility of a more important role for this complex under flow, rather than static conditions.

Exposure to shear stress has been shown to alter the expression of a number of surface components of cultured endothelial cells (EC). However, relatively few studies have examined the status of human EC surface proteins after prolonged flow, more closely corresponding to the steady state in vivo. Since the promoter region of glycoprotein (Gp) Ib alpha contains several copies of a putative shear stress response element, 5'-GAGACC-3', we investigated the response of cultured human umbilical vein EC (HUVEC) GpIb alpha to shear stress over a 72 h time period. In response to 30 dynes/cm2 of shear stress, total cell content of GpIb alpha protein was markedly increased above static levels at 7 and 24 h, as determined immunohistochemically. Western blot analysis of whole cell lysates after 24, 48, and 72 h of shear treatment demonstrated a 2.4-, 4.1-, and 3.2-fold increase in total GpIb alpha protein, respectively. Cell surface protein expression of GpIb alpha increased 2.5-fold at 7 h, as measured by quantitative immunofluorescence, and remained at that level at 24 h. After 48 h of shear stress, cell surface GpIb alpha, GpIX, and GpV, analyzed by flow cytometric analysis, were further increased over the levels observed at 24 h. The increase in cell surface membrane expression of GPIb alpha at 24, 48, and 72 h was confirmed by immunoprecipitation of biotinylated surface proteins. No upregulation of GpIb alpha was noted after exposure to shear stress of 1-3 dynes/cm2. These observations imply that under steady-state arterial shear conditions endothelial expression of the GpIb complex is significantly greater than observed in static EC cultures, and raise the possibility of a more important role for this complex under flow, rather than static conditions.