The small GTP-binding protein Rho has
been implicated in the control of neuronal morphology.
In N1E-115 neuronal cells, the Rho-inactivating C3
toxin stimulates neurite outgrowth and prevents actomyosin-based neurite retraction and cell rounding
induced by lysophosphatidic acid (LPA), sphingosine-1-phosphate, or thrombin acting on their cognate G
protein–coupled receptors. We have identified a novel
putative GDP/GTP exchange factor, RhoGEF (190
kD), that interacts with both wild-type and activated
RhoA, but not with Rac or Cdc42. RhoGEF, like activated RhoA, mimics receptor stimulation in inducing
cell rounding and in preventing neurite outgrowth. Furthermore, we have identified a 116-kD protein, p116Rip,
that interacts with both the GDP- and GTP-bound
forms of RhoA in N1E-115 cells. Overexpression of
p116Rip stimulates cell flattening and neurite outgrowth
in a similar way to dominant-negative RhoA and C3
toxin. Cells overexpressing p116Rip fail to change their
shape in response to LPA, as is observed after Rho inactivation. Our results indicate that (a) RhoGEF may
link G protein–coupled receptors to RhoA activation
and ensuing neurite retraction and cell rounding; and
(b) p116Rip inhibits RhoA-stimulated contractility and
promotes neurite outgrowth.
Diacylglycerol kinase (DGK) attenuates levels of second messenger diacylglycerol in cells and produces another (putative) messenger, phosphatidic acid. We have previously purified a 110-kDa DGK from rat brain (Kato, M., and Takenawa, T. (1990) J. Biol. Chem. 265, 794 -800). Here we report the cDNA cloning from human brain and retina cDNA libraries. The cDNA encodes a novel DGK isotype, termed DGK, of 941 amino acids with an apparent molecular mass of 110 kDa. DGK contains a C-terminal putative catalytic domain, which is present in all eukaryotic DGKs. In contrast to other DGK isotypes, DGK contains three cysteine-rich domains instead of two. The third cysteine-rich domain is most homologous to the second one in other DGK isotypes. This particular sequence homology extends C-terminally beyond the typical cysteine/histidine core structure and is DGKspecific. DGK furthermore contains various domains for protein-protein interaction, such as a proline-and glycine-rich domain with a putative SH3 domain-binding site and a pleckstrin homology domain with an overlapping Ras-associating domain. DGK is expressed in the brain and, to a lesser extent, in the small intestine, duodenum, and liver. In situ hybridization of DGK mRNA in adult rat brain reveals high expression in the cerebellar cortex and hippocampus. DGK activity in COS cell lysates is optimal toward diacylglycerols containing an unsaturated fatty acid at the sn-2 position.
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