H-Ras, R-Ras, and TC21 differentially regulate ureteric bud cell branching morphogenesis.

The collecting system of the kidney, derived from the ureteric bud (UB), undergoes repetitive bifid branching events during early development followed by a phase of tubular growth and elongation. Although members of the Ras GTPase family control cell growth, differentiation, proliferation, and migration, their role in development of the collecting system of the kidney is unexplored. In this study, we demonstrate that members of the R-Ras family of proteins, R-Ras and TC21, are expressed in the murine collecting system at E13.5, whereas H-Ras is only detected at day E17.5. Using murine UB cells expressing activated H-Ras, R-Ras, and TC21, we demonstrate that R-Ras-expressing cells show increased branching morphogenesis and cell growth, TC21-expressing cells branch excessively but lose their ability to migrate, whereas H-Ras-expressing cells migrated the most and formed long unbranched tubules. These differences in branching morphogenesis are mediated by differential regulation/activation of the Rho family of GTPases and mitogen-activated protein kinases. Because most branching of the UB occurs early in development, it is conceivable that R-Ras and TC-21 play a role in facilitating branching and growth in early UB development, whereas H-Ras might favor cell migration and elongation of tubules, events that occur later in development.

CD98 modulates integrin beta1 function in polarized epithelial cells.

The type II transmembrane protein CD98, best known as the heavy chain of the heterodimeric amino acid transporters (HAT), is required for the surface expression and basolateral localization of this transporter complex in polarized epithelial cells. CD98 also interacts with beta1 integrins resulting in an increase in their affinity for ligand. In this study we explored the role of the transmembrane and cytoplasmic domains of CD98 on integrin-dependent cell adhesion and migration in polarized renal epithelial cells. We demonstrate that the transmembrane domain of CD98 was sufficient, whereas the five N-terminal amino acids of this domain were required for CD98 interactions with beta1 integrins. Overexpression of either full-length CD98 or CD98 lacking its cytoplasmic tail increased cell adhesion and migration, whereas deletion of the five N-terminal amino acids of the transmembrane domain of CD98 abrogated this effect. CD98 and mutants that interacted with beta1 integrins increased both focal adhesion formation and FAK and AKT phosphorylation. CD98-induced cell adhesion and migration was inhibited by addition of phosphoinositol 3-OH kinase (PI3-K) inhibitors suggesting these cell functions are PI3-K-dependent. Finally, CD98 and mutants that interacted with beta1, induced marked changes in polarized renal epithelial cell branching morphogenesis in collagen gels. Thus, in polarized renal epithelial cells, CD98 might be viewed as a scaffolding protein that interacts with basolaterally expressed amino acid transporters and beta1 integrins and can alter diverse cellular functions such as amino acid transport as well as cell adhesion, migration and branching morphogenesis.

Transport of 5-formyltetrahydrofolate into primary cultured rat astrocytes.

Transport of 5-formyltetrahydrofolate (5-FTHF) into primary cultured rat astrocytes was studied. Uptake of 5-FTHF into astrocytes was linear in the first 60 min and is saturable with K(m)=3.3 microM and V(max)=27.5 pmol/mg protein/45 min in pH 7.4 medium. Uptake of 5-FTHF displayed the characteristics of countertransport. Uptake of 5-FTHF was inhibited by the structural analogs 5-methyltetrahydrofolate, methtrexate, and folic acid (K(i)=3.8, 2.7, and 18.4 microM, respectively). Uptake was significantly decreased by sodium azide but was increased by high concentration of sodium cyanide and low concentration of sodium arsenate. Uptake was also inhibited by p-chloromercuriphenylsulfonate and by the anions probenecid and 4,4(')-diisothiocyanostilbene-2,2(')-disulfonic acid. Acute exposure of the cells to ethanol (100mM) inhibited the uptake for 90 min of the experimental duration. It is concluded that astrocytes have a system for the uptake of 5-FTHF and folate analogs which is carrier mediated, this system is sensitive to energy inhibitors and alcohol exposure.

Transport of 5-formyltetrahydrofolate into primary cultured cerebellar granule cells.

Transport of 5-formyltetrahydrofolate (5-FTHF) into primary cultured cerebellar granule cells (CGC) was studied. Uptake of 5-FTHF into CGC was saturable with K(m)=2.86 microM and V(max)=40.8 pmol/mg protein/45 min in pH 7.4 medium. Uptake of 5-FTHF in the astrocytes has a similar style in the time curve. Uptake of 5-FTHF is characterized by countertransport because adding unlabeled 5-FTHF in the medium resulted in the efflux of labeled 5-FTHF. Uptake of 5-FTHF was inhibited by the structural analogs 5-methyltetrahydrofolate, methotrexate and folic acid (K(i)=6.64, 7.69, and 19.38 microM, respectively). Uptake was significantly decreased by high concentrations of sodium azide and sodium arsenate but not by sodium cyanide. Uptake was also inhibited by p-chloromercuriphenylsulfonate and by the anions probenecid and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Acute exposure of the cells to ethanol (100 mM) did not affect the uptake. It is concluded that CGC have a carrier-mediated system for the uptake of 5-FTHF and other folates.

Mechanisms of regulation of phospholipase D1 and D2 by the heterotrimeric G proteins G13 and Gq.

Our earlier studies of rat brain phospholipase D1 (rPLD1) showed that the enzyme could be activated in cells by alpha subunits of the heterotrimeric G proteins G(13) and G(q). Recently, we showed that rPLD1 is modified by Ser/Thr phosphorylation and palmitoylation. In this study, we first investigated the roles of these post-translational modifications on the activation of rPLD1 by constitutively active Galpha(13)Q226L and Galpha(q)Q209L. Mutations of Cys(240) and Cys(241) of rPLD1, which abolish both post-translational modifications, did not affect the ability of either Galpha(13)Q226L or Galpha(q)Q209L to activate rPLD1. However, the RhoA-insensitive mutants, rPLD1(K946A,K962A) and rPLD1(K962Q), were not activated by Galpha(13)Q226L, although these mutant enzymes responded to phorbol ester and Galpha(q)Q209L. On the contrary, the PKC-insensitive mutant rPLD1(DeltaN168), which lacks the first 168 amino acids of rPLD1, responded to Galpha(13)Q226L but not to Galpha(q)Q209L. In addition, we found that rPLD2 was strongly activated by Galpha(q)Q209L and phorbol ester. However, surprisingly, the enzymatic activity of rPLD2 was suppressed by Galpha(13)Q226L and constitutively active V14RhoA in COS-7 cells. Abolition of the post-translational modifications of rPLD2 did not alter the effects of Galpha(q)Q209L or Galpha(13)Q226L. The suppressive effect of Galpha(13)Q226L on rPLD2 was reversed by dominant negative N19RhoA and the C3 exoenzyme of Clostridium botulinum, further supporting a role for RhoA. In summary, Galpha(13) activation of rPLD1 in COS-7 cells is mediated by Rho, while Galpha(q) activation requires PKC. rPLD2 is activated by Galpha(q), but is inhibited by Galpha(13). Neither Ser/Thr phosphorylation nor palmitoylation is required for these effects.