Insulin stimulates glucose uptake into its target cells by a process which involves the translocation of the GLUT4 isoform of glucose transporter from an intracellular vesicular compartment(s) to the plasma membrane. The step(s) at which insulin acts in the vesicle trafficking pathway (e.g. vesicle movement or fusion with the plasma membrane) is not known. We expressed a green-fluorescent protein-GLUT4 (GFP-GLUT4) chimaera in 3T3 L1 adipocytes. The chimaera was expressed in vesicles located throughout the cytoplasm and also close to the plasma membrane. Insulin promoted a substantial translocation of GFP-GLUT4 to the plasma membrane. Time-lapse confocal microscopy demonstrated that the majority of GFP-GLUT4-containing vesicles in the basal state were relatively static, as if tethered (or attached) to an intracellular structure. A proportion (approx. 5%) of the vesicles spontaneously lost their tether, and were observed to move rapidly within the cell. Other vesicles appear to be tethered only on one edge and were observed in a rapid stretching motion. The data support a model in which GLUT4-containing vesicles are tightly tethered to an intracellular structure(s), and indicate that a primary site of insulin action must be to release these vesicles, allowing them to then translocate to and fuse with the plasma membrane.

Regulation of phosphoinositide 3-kinase (PI 3-kinase) can occur by binding of the regulatory p85 subunit to tyrosine-phosphorylated proteins and by binding of the p110 catalytic subunit to activated Ras. However, the way in which these regulatory mechanisms act to regulate PI 3-kinase in vivo is unclear. Here we show that several growth factors (basic fibroblast growth factor [bFGF], platelet-derived growth factor [PDGF], and epidermal growth factor [EGF; to activate an EGF receptor-Ret chimeric receptor]) all activate PI 3-kinase in vivo in the neuroectoderm-derived cell line SKF5. However, these growth factors differ in their ability to activate PI 3-kinase-dependent signaling. PDGF and EGF(Ret) treatment induced PI 3-kinase-dependent lamellipodium formation and protein kinase B (PKB) activation. In contrast, bFGF did not induce lamellipodium formation but activated PKB, albeit to a small extent. PDGF and EGF(Ret) stimulation resulted in binding of p85 to tyrosine-phosphorylated proteins and strong Ras activation. bFGF, however, induced only strong activation of Ras. In addition, while Ras Asn17 abolished bFGF activation of PKB, PDGF-and EGF(Ret)-induced PKB activation was only partially inhibited and lamellipodium formation was unaffected. Interestingly, in contrast to activation of only endogenous Ras (bFGF), ectopic expression of activated Ras did result in lamellipodium formation. From this we conclude that, in vivo, p85 and Ras synergize to activate PI 3-kinase and that strong activation of only endogenous Ras exerts a small effect on PI 3-kinase activity, sufficient for PKB activation but not lamellipodium formation. This differential sensitivity to PI 3-kinase activation could be explained by our finding that PKB activation and lamellipodium formation are independent PI 3-kinaseinduced events.After growth factor binding to a receptor tyrosine kinase, a variety of signaling events are induced. Among these are the activation of the small GTPase Ras and the activation of phosphoinositide 3-kinase (PI 3-kinase). The active GTP-bound form of Ras binds to several effector molecules, including (i) family members of the serine-threonine kinase Raf1, which results in the activation of the kinases extracellular signalregulated kinase (ERK) and mitogen-activated protein/ERK kinase (MEK) (29); (ii) Ral guanine nucleotide exchange factor (RalGEF) family members, which leads to the activation of the small GTPase Ral (15); and, interestingly, (iii) members of the PI 3-kinase family (35). PI 3-kinase phosphorylates phosphatidylinositol (PI) lipids on the 3Ј position of the inositol ring, and growth factor-induced PI 3-kinase activation leads to an increase in the levels of PI-3,4,5 triphosphate (PI-3,4,5P 3 ) and PI-3,4P 2 (21, 41). PI 3-kinase has been implicated in a large variety of biological responses to growth factors; these include actin reorganization (lamellipodium formation), apoptosis protection, glucose uptake, activation of kinases (p70 s6k , protein kinase B [PKB], and atypical PKCs), and inactivation of ...

Ret is a receptor tyrosine kinase required during embryogenesis for the survival of enteric and sympathetic neuroblasts. Recently, glial cell line-derived neurotrophic factor (GDNF) has been identified as a ligand for Ret. We investigated early events in GDNF-induced signal transduction. We show that GDNF activates the Ras-ERK2 signaling pathway in SK-N-MC cells stably transfected with a full-length Ret construct. In addition, activation of Ret tyrosine kinase activity, either via GDNF stimulation of full-length Ret or via epidermal growth factor stimulation of an epidermal growth factor receptor-Ret chimeric receptor, results in phosphatidylinositol 3-kinase activation and phosphatidylinositol 3-kinase-dependent formation of large lamellipodia. Our results indicate that GDNF can serve as a genuine ligand for Ret. In addition, we show that GDNF can induce Ret-mediated formation of lamellipodia, cell surface protrusions that are implicated in neuritogenesis.

CD44 is described to be an activation molecule in a number of different cell types. We investigated the role of CD44 on human endothelial cells (EC) and in tumor angiogenesis. Using flow cytometry we showed that EC from the vasculature of human solid tumors display an enhanced expression of CD44 as compared to EC from normal tissue. This finding was confirmed by immunohistochemical studies on frozen tissue sections. Because tumors are dependent on angiogenesis, the role of angiogenic stimuli in the enhanced CD44 expression was investigated. We found that basic fibroblast growth factor (bFGF ) and vascular endothelial growth factor were able to efficiently upregulate CD44 expression on cultured human EC. The upregulation reached maximal levels after treatment for 3 days with 10 ng/mL bFGF. The physiological impact of this upregulation was shown by the enhanced binding of EC to hyaluronate after pretreatment with bFGF. In a next set of studies that were designed to unravel the regulation of CD44 expression on EC we concluded that CD44 is an activation antigen on human EC since (1) human umbilical vein derived endothelial cells, which in vivo do not express CD44, begin to express CD44 when plated and cultured, (2) CD44 expression is enhanced after subculture of confluent cultures, (3) CD44 is predominantly expressed on the BrdU incorporating subset of cultured EC. The specific expression of CD44 on activated and tumor EC prompted us to study the usefulness of CD44 as an endothelial target for therapy with immunotoxins. In vitro experiments showed that EC are efficiently killed after targeting immunotoxin to CD44.

No abstract

Mutations in the Ret receptor tyrosine kinase are responsible for a variety of human syndromes, including multiple endocrine neoplasia 2 and Hirschsprung's disease. Ret is expressed as a 150-kDa precursor form in the endoplasmic reticulum and a 170-kDa mature form at the plasma membrane. Here we show that expression of p170 ret is dependent on calcium. Depletion of extracellular calcium completely blocks p170 ret expression, which is not caused by a decrease in half-life of p170 ret at the plasma membrane but by a defect in processing of p150 ret into p170 ret . This processing defect can be mimicked by treating the cells with thapsigargin, a drug that releases calcium from internal stores, indicating that reduction in luminal calcium is responsible for the processing defect. We propose that a relatively high concentration of luminal calcium is necessary for the proper folding of Ret in the endoplasmic reticulum.

CD44 is described to be an activation molecule in a number of different cell types. We investigated the role of CD44 on human endothelial cells (EC) and in tumor angiogenesis. Using flow cytometry we showed that EC from the vasculature of human solid tumors display an enhanced expression of CD44 as compared to EC from normal tissue. This finding was confirmed by immunohistochemical studies on frozen tissue sections. Because tumors are dependent on angiogenesis, the role of angiogenic stimuli in the enhanced CD44 expression was investigated. We found that basic fibroblast growth factor (bFGF ) and vascular endothelial growth factor were able to efficiently upregulate CD44 expression on cultured human EC. The upregulation reached maximal levels after treatment for 3 days with 10 ng/mL bFGF. The physiological impact of this upregulation was shown by the enhanced binding of EC to hyaluronate after pretreatment with bFGF. In a next set of studies that were designed to unravel the regulation of CD44 expression on EC we concluded that CD44 is an activation antigen on human EC since (1) human umbilical vein derived endothelial cells, which in vivo do not express CD44, begin to express CD44 when plated and cultured, (2) CD44 expression is enhanced after subculture of confluent cultures, (3) CD44 is predominantly expressed on the BrdU incorporating subset of cultured EC. The specific expression of CD44 on activated and tumor EC prompted us to study the usefulness of CD44 as an endothelial target for therapy with immunotoxins. In vitro experiments showed that EC are efficiently killed after targeting immunotoxin to CD44.