The GRP1 protein contains a Sec7 homology domain that catalyzes guanine nucleotide exchange on ADP-ribosylation factors (ARF) 1 and 5 as well as a pleckstrin homology domain that binds phosphatidylinositol-(3,4,5)P 3 , an intermediate in cell signaling by insulin and other extracellular stimuli (Klarlund, J. K., Guilherme, A., Holik, J. J., Virbasius, J. V., Chawla, A., and Czech, M. P. (1997) Science 275, 1927Science 275, -1930). Here we show that both endogenous GRP1 and ARF6 rapidly co-localize in plasma membrane ruffles in Chinese hamster ovary (CHO-T) cells expressing human insulin receptors and COS-1 cells in response to insulin and epidermal growth factor, respectively. The pleckstrin homology domain of GRP1 appears to be sufficient for regulated membrane localization. Using a novel method to estimate GTP loading of expressed HA epitope-tagged ARF proteins in intact cells, levels of biologically active, GTP-bound ARF6 as well as GTP-bound ARF1 were elevated when these ARF proteins were co-expressed with GRP1 or the related protein cytohesin-1. GTP loading of ARF6 in both control cells and in response to GRP1 or cytohesin-1 was insensitive to brefeldin A, consistent with previous data on endogenous ARF6 exchange activity. The ability of GRP1 to catalyze GTP/GDP exchange on ARF6 was confirmed using recombinant proteins in a cell-free system. Taken together, these results suggest that phosphatidylinositol-(3,4,5)P 3 may be generated in cell membrane ruffles where receptor tyrosine kinases are concentrated in response to growth factors, causing recruitment of endogenous GRP1. Further, co-localization of GRP1 with ARF6, combined with its demonstrated ability to activate ARF6, suggests a physiological role for GRP1 in regulating ARF6 functions.Signaling through receptor tyrosine kinases regulates multiple processes critical to cell growth, differentiation, and viability (1-3). A common element of receptor tyrosine kinase signaling that is required to regulate many cellular processes appears to be a class of enzymes, PI 1 3-kinases (4 -6), that catalyze phosphorylation of the 3Ј-position on phosphatidylinositol (PtdIns) or its phosphorylated derivatives to produce PtdIns(3)P, PtdIns(3,4)P 2 , or PtdIns(3,4,5)P 3 . We recently identified a protein, GRP1, that binds PtdIns(3,4,5)P 3 with high affinity through its pleckstrin homology (PH) domain and contains a Sec7 homology domain that catalyzes guanine nucleotide exchange of ADP-ribosylation factor (ARF) proteins (7,8). Two isoforms of GRP1 denoted ARNO (9) and cytohesin-1 (10) with similar domain structures have also been identified. GRP1, ARNO, and cytohesin-1 expressed in cultured cells as fusion proteins of green fluorescent protein are rapidly recruited to the plasma membrane in response to receptor tyrosine kinase activation (11-13). Such recruitment appears to require the PH domains of these proteins. However, neither the cellular localization nor the regulation of endogenous GRP1-like proteins has yet been characterized. ARF proteins appear to regulate me...

Insulin regulates glucose uptake in adipocytes and muscle by stimulating the movement of sequestered glucose transporter 4 (GLUT4) proteins from intracellular membranes to the cell surface. Here we report that optimal insulin-mediated GLUT4 translocation is dependent upon both microtubule and actin-based cytoskeletal structures in cultured adipocytes. Depolymerization of microtubules and F-actin in 3T3-L1 adipocytes causes the dispersion of perinuclear GLUT4-containing membranes and abolishes insulin action on GLUT4 movements to the plasma membrane. Furthermore, heterologous expression in 3T3-L1 adipocytes of the microtubule-binding protein hTau40, which impairs kinesin motors that move toward the plus ends of microtubules, markedly delayed the appearance of GLUT4 at the plasma membrane in response to insulin. The hTau40 protein had no detectable effect on microtubule structure or perinuclear GLUT4 localization under these conditions. These results are consistent with the hypothesis that both the actin and microtubule-based cytoskeleton, as well as a kinesin motor, direct the translocation of GLUT4 to the plasma membrane in response to insulin.Physiological glucose homeostasis in humans is largely dependent on the actions of the hormone insulin, particularly its ability to inhibit glucose output from the liver and enhance glucose transport into fat and muscle cells. Insulin exerts this latter effect primarily through a process whereby sequestered intracellular GLUT4 glucose transporter proteins are rapidly redistributed to cell surface membranes in which they can catalyze glucose uptake into cells (1-4). In both the basal and insulin-stimulated states, GLUT4 proteins appear to cycle between intracellular membrane and plasma membrane locations (5). However, in the basal state most of the GLUT4 is diverted to intracellular perinuclear membranes, and the exocytosis rate is slow. Insulin stimulates exocytosis of GLUT4 by a mechanism that requires the p85/p110 type phosphatidylinositol 3-kinase, which is recruited to protein phosphotyrosines in response to activation of the insulin receptor tyrosine kinase (6 -8). Insulin also appears to significantly inhibit GLUT4 endocytosis (5, 9, 10). However, the precise mechanism whereby insulin signals to the GLUT4 membrane trafficking machinery remains obscure.It is known that other membrane systems, such as lysosomes (11), mitochondria (12), Golgi membranes (13, 14), and pigment granules (15), are localized within cells by molecular motors. For example, membrane vesicles containing melanin appear to be driven along microtubules over relatively long distances from their perinuclear location in unstimulated melanocytes to the cell periphery upon elevation of cAMP levels (16). The complex motor dynein drives movements along microtubules in the minus direction toward the perinuclear microtubule organizing center, whereas kinesin motors drive movements toward the plus growing ends of microtubules (17). Movements of these membranes over shorter distances seem to require actin fila...

Clinicians have had concerns about particulate matter contamination of injectable drug products since the development of the earliest intravenous therapeutics. All parenteral products contain particulate matter, and particulate matter contamination still has the potential to cause harm to patients. With tens of millions of doses of injectable drug products administered in the United States each year, it is critical to understand the types and sources of particulate matter that contaminate injectable drug products, the possible effects of injected particulate matter on patients, and the current state of regulations and standards related to particulate matter in injectable drug products. Today, the goal of manufacturers, regulators, and standards-setting organizations should be to continue to minimize the risk of particle-induced sequelae, especially in high-risk patients, without trading unnecessary manufacturing burden for minimal safety gains.

The action of insulin to recruit the intracellular GLUT4 glucose transporter to the plasma membrane of 3T3-L1 adipocytes is mimicked by endothelin 1, which signals through trimeric G ␣ q or G ␣ 11 proteins. Here we report that murine G ␣ 11 is most abundant in fat and that expression of the constitutively active form of G ␣ 11 [G ␣ 11(Q209L)] in 3T3-L1 adipocytes causes recruitment of GLUT4 to the plasma membrane and stimulation of 2-deoxyglucose uptake. In contrast to the action of insulin on GLUT4, the effects of endothelin 1 and G ␣ 11 were not inhibited by the phosphatidylinositol 3-kinase inhibitor wortmannin at 100 nM. Signaling by insulin, endothelin 1, or G ␣ 11(Q209L) also mobilized cortical F-actin in cultured adipocytes. Importantly, GLUT4 translocation caused by all three agents was blocked upon disassembly of F-actin by latrunculin B, suggesting that the F-actin polymerization caused by these agents may be required for their effects on GLUT4. Remarkably, expression of a dominant inhibitory form of the actin-regulatory GTPase ARF6 [ARF6(T27N)] in cultured adipocytes selectively inhibited both F-actin formation and GLUT4 translocation in response to endothelin 1 but not insulin. These data indicate that ARF6 is a required downstream element in endothelin 1 signaling through G ␣ 11 to regulate cortical actin and GLUT4 translocation in cultured adipocytes, while insulin action involves different signaling pathways.

There has been much written on bacterial exopolysaccharides (EPS) and their role in virulence. Less has been published regarding EPS in free living species. This review focuses on that subject, emphasizing their functions in the environment and the use of antibody probes to study them.