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The apical junctional complex (AJC) consists of adherens junctions (AJs) and tight junctions and regulates epithelial integrity and remodeling. However, it is unclear how AJC organization is regulated based on environmental cues. We found here using cultured EpH4 mouse mammary epithelial cells that fetal bovine serum (FBS) in a culture medium showed an activity to promote AJC organization and that FBS showed an activity to promote tight junction formation even in the absence of AJ proteins, such as E-cadherin, αE-catenin, and afadin. Furthermore, we purified the individual factor responsible for these functions from FBS and identified this molecule as lysophosphatidic acid (LPA). In validation experiments, purified LPA elicited the same activity as FBS. In addition, we found that the AJC organization–promoting activity of LPA was mediated through the LPA receptor 1/5 via diacylglycerol–novel PKC and Rho–ROCK pathway activation in a mutually independent, but complementary, manner. We demonstrated that the Rho–ROCK pathway activation–mediated AJC organization was independent of myosin II-induced actomyosin contraction, although this signaling pathway was previously shown to induce myosin II activation. These findings are in contrast to the literature, as previous results suggested an AJC organization–disrupting activity of LPA. The present results indicate that LPA in serum has an AJC organization–promoting activity in a manner dependent on or independent of AJ proteins.

During epithelial morphogenesis, adherens junctions (AJs) and tight junctions (TJs) undergo dynamic reorganization, whereas epithelial polarity is transiently lost and reestablished. Although ARF6-mediated endocytic recycling of E-cadherin has been characterized and implicated in the rapid remodeling of AJs, the molecular basis for the dynamic rearrangement of TJs remains elusive. Occludin and claudins are integral membrane proteins comprising TJ strands and are thought to be responsible for establishing and maintaining epithelial polarity. Here we investigated the intracellular transport of occludin and claudins to and from the cell surface. Using cell surface biotinylation and immunofluorescence, we found that a pool of occludin was continuously endocytosed and recycled back to the cell surface in both fibroblastic baby hamster kidney cells and epithelial MTD-1A cells. Biochemical endocytosis and recycling assays revealed that a Rab13 dominant active mutant (Rab13 Q67L) inhibited the postendocytic recycling of occludin, but not that of transferrin receptor and polymeric immunoglobulin receptor in MTD-1A cells. Double immunolabelings showed that a fraction of endocytosed occludin was colocalized with Rab13 in MTD-1A cells. These results suggest that Rab13 specifically mediates the continuous endocytic recycling of occludin to the cell surface in both fibroblastic and epithelial cells.

Rab11 small G protein has been implicated in vesicle recycling, but its upstream regulators or downstream targets have not yet been identified. We isolated here a downstream target of Rab11, named rabphilin-11, from bovine brain. Moreover, we isolated from a rat brain cDNA library its cDNA, which encoded a protein with a M(r) of 100,946 and 908 amino acids (aa). Rabphilin-11 bound GTP-Rab11 more preferentially than GDP-Rab11 at the N-terminal region and was specific for Rab11 and inactive for other Rab and Rho small G proteins. Both GTP-Rab11 and rabphilin-11 were colocalized at perinuclear regions, presumably the Golgi complex and recycling endosomes, in Madin-Darby canine kidney cells. In HeLa cells cultured on fibronectin, both the proteins were localized not only at perinuclear regions but also along microtubules, which were oriented toward membrane lamellipodia. Treatment of HeLa cells with nocodazole caused disruption of microtubules and dispersion of GTP-Rab11 and rabphilin-11. Overexpression of the C-terminal fragment of rabphilin-11 (aa 607-730), lacking the GTP-Rab11 binding domain, in HeLa cells reduced accumulation of transferrin at perinuclear regions and cell migration. Rabphilin-11 turned out to be a rat counterpart of recently reported bovine Rab11BP. These results indicate that rabphilin-11 is a downstream target of Rab11 which is involved in vesicle recycling.

The Rab3 small G protein subfamily (Rab3) consists of four members, Rab3A, -B, -C, and -D. We have recently isolated and characterized the Rab3 regulators, GDP/GTP exchange protein (GEP) and GTPase activating protein (GAP), both of which are specific for the Rab3 subfamily. Rab3 GEP stimulates the conversion of the GDP-bound inactive form to the GTP-bound active form, whereas Rab3 GAP stimulates the reverse reaction. Of the four members of the Rab3 subfamily, evidence is accumulating that Rab3A is involved in Ca2+-dependent exocytosis, particularly in neurotransmitter release. We first analyzed the subcellular localization of Rab3 GEP and GAP in rat brain. Subcellular fractionation analysis showed that both Rab3 GEP and GAP were enriched in the synaptic soluble fraction. Immunocytochemical analysis in primary cultured rat hippocampal neurons showed that both Rab3 GEP and GAP were concentrated at the presynaptic nerve terminals. We then examined whether Rab3 GEP and GAP were involved in Ca2+-dependent exocytosis by use of human growth hormone (GH) co-expression assay system of cultured PC12 cells. Overexpression of the deletion mutant of Rab3 GEP possessing the catalytic activity reduced the high K+-induced GH release without affecting the basal GH release, whereas that of the deletion mutant lacking the catalytic activity showed no effect on the high K+-induced GH release. In contrast, overexpression of Rab3 GAP or its deletion mutant possessing the catalytic activity did not affect the high K+-induced GH release or the basal GH release. These results indicate that Rab3 GEP and GAP are colocalized with Rab3A at the synaptic release sites and suggest that they regulate the activity of Rab3A and are involved in Ca2+-dependent exocytosis.

Rabphilin3 and rabaptin5 are downstream target molecules of the Rab3 and -5 subfamily small G proteins that are implicated in exocytosis and endocytosis, respectively. We examined here the physical and functional relationship between the Rab3-rabphilin3 and Rab5-rabaptin5 systems. Rabphilin3 interacted with rabaptin5 at the N-terminal region (amino acids 1–280), which GTP-Rab3A interacted with. The interaction of rabphilin3 with rabaptin5 was inhibited by guanosine 5′-(3-O-thio)triphosphate-Rab3A. Overexpression of the N-terminal fragment of rabphilin3 (amino acids 1–280) inhibited the receptor-mediated endocytosis of transferrin, and this inhibition was overcome by co-transfection with a dominant active mutant of Rab3A or rabaptin5 in PC12 and HeLa cells. These results suggest that rabphilin3, free of GTP-Rab3A, regulates endocytosis through interaction with rabaptin5 after rabphilin3 complexed with GTP-Rab3A regulates exocytosis.

The Rho GDP dissociation inhibitor (GDI) forms a complex with the GDP-bound form of the Rho family small G proteins and inhibits their activation. The GDP-bound form complexed with Rho GDI is not activated by the GDP/GTP exchange factor for the Rho family members, suggesting the presence of another factor necessary for this activation. We have reported that the Rho subfamily members regulate the ezrin/radixin/moesin (ERM)-CD44 system, implicated in reorganization of actin filaments. Here we report that Rho GDI directly interacts with ERM, initiating the activation of the Rho subfamily members by reducing the Rho GDI activity. These results suggest that ERM as well as Rho GDI and the Rho GDP/GTP exchange factor are involved in the activation of the Rho subfamily members, which then regulate reorganization of actin filaments through the ERM system.

Doc2 has two C2 domains that interact with Ca2+ and phospholipid. Munc13 has two C2 domains and one C1 domain that interacts with phorbol ester or diacylglycerol (DAG) and phospholipid. Both Doc2 and Munc13 are implicated in Ca2+-dependent neurotransmitter release, but their modes of action still remain unclear. We show here that Doc2 interacts with Munc13 both in a cell-free system and in intact PC12 cells during the high K+-induced Ca2+-dependent exocytosis. The Doc2-Munc13 interactions are stimulated by phorbol ester through the C1 domain of Munc13. Overexpression of the Doc2-interacting domain of Munc13 reduces the Ca2+-dependent exocytosis from PC12 cells, and co-expression with Doc2 suppresses this reduction. These results, together with the earlier findings that secretagogues produce DAG and elevate cytoplasmic Ca2+, suggest that the DAG-induced Doc2-Munc13 interactions play an important role in Ca2+-dependent exocytotic machinery.

The Rab small G protein family, consisting of nearly 30 members, is implicated in intracellular vesicle trafficking. They cycle between the GDP-bound and GTP-bound forms, and the latter is converted to the former by the action of a GTPase activating protein (GAP). No GAP specific for each Rab family member or Rab subfamily has been isolated in mammal. Here we purified a GAP with Rab3A as a substrate from rat brain. The purified protein was specifically active on the Rab3 subfamily members (Rab3A, -B, -C, and -D). Of this subfamily, Rab3A and -C are implicated in Ca2+-dependent exocytosis, particularly in neurotransmitter release. This GAP, named Rab3 GAP, was active on the lipid-modified form, but not on the lipid-unmodified form. Rab3 GAP showed a minimum molecular mass of about 130 kDa on SDS-polyacrylamide gel electrophoresis. We cloned its cDNA from a human brain cDNA library, and the isolated cDNA encoded a protein with a Mr of 110,521 and 981 amino acids, which showed no homology to any known protein. The recombinant protein exhibited GAP activity toward the Rab3 subfamily members, and the catalytic domain was located at the C-terminal region. Northern blot analysis indicated that Rab3 GAP was ubiquitously expressed.

Rabphilin-3A is a downstream target molecule of Rab3A small GTP-binding protein and implicated in Ca2+-dependent neurotransmitter release. Here we have isolated a rabphilin-3A-interacting molecule from a human brain cDNA library by the yeast two-hybrid method and identified it to be alpha-actinin, known to cross-link actin filaments into a bundle. alpha-Actinin interacts with the N-terminal region of rabphilin-3A, with which GTP-Rab3A interacts, and this interaction stimulates the activity of alpha-actinin to cross-link actin filaments into a bundle. The interaction of rabphilin-3A with alpha-actinin is inhibited by guanosine 5'-(3-O-thio)triphosphate-Rab3A. These results suggest that the Rab3A-rabphilin-3A system regulates the alpha-actinin-regulated reorganization of actin filaments. It has been shown that reorganization of actin filaments is also involved in Ca2+-dependent exocytosis. Therefore, rabphilin-3A may serve as a linker for Rab3A and cytoskeleton.

An activator of rat brain phospholipase D (PLD) that is distinct from the already identified PLD activator, ADP-ribosylation factor (ARF), was partially purified from bovine brain cytosol by a series of chromatographic steps. The partially purified preparation contained a 22-kDa substrate for Clostridium botulinum C3 exoenzyme ADP-ribosyltransferase, which strongly reacted with anti-rhoA p21 antibody, but not with anti-rac1 p21 or anti-cdc42Hs p21 antibody. Treatment of the partially purified PLD-activating factor with both C3 exoenzyme and NAD significantly inhibited the PLD-stimulating activity. These results suggest that rhoA p21 is, at least in part, responsible for the PLD-stimulating activity in the preparation. Recombinant isoprenylated rhoA p21 expressed in and purified from Sf9 cells activated rat brain PLD in a concentration- and GTP gamma S (guanosine 5'-O-(3-thiotriphosphate))-dependent manner. In contrast, recombinant non-isoprenylated rhoA p21 (fused to glutathione S-transferase) expressed in Escherichia coli failed to activate the PLD. This difference cannot be explained by a lower affinity of non-isoprenylated rhoA p21 for GTP gamma S, as the rates of [35S]GTP gamma S binding were very similar for both recombinant preparations and the GTP gamma S-bound form of non-isoprenylated rhoA p21 did not induce PLD activation. Interestingly, recombinant isoprenylated rhoA p21 and ARF synergistically activated rat brain PLD; a similar pattern was seen with the partially purified PLD-activating factor. The synergistic activation was inhibited by C3 exoenzyme-catalyzed ADP-ribosylation of recombinant isoprenylated rhoA p21 in a NAD-dependent manner. Inhibition correlated with the extent of ADP-ribosylation. These findings suggest that rhoA p21 regulates rat brain PLD in concert with ARF, and that isoprenylation of rhoA p21 is essential for PLD regulation in vitro.

rho GDP dissociation inhibitor (GDI) is an inhibitory GDP/GTP exchange protein for a group of small GTP-binding proteins including at least rhoA p21, rhoB p21, rac1 p21, rac2 p21, and G25K. Microinjection of rho GDI into Swiss 3T3 cells made the cells round and refractile. This morphological change was accompanied by the disappearance of stress fibers. The rho GDI action was prevented by comicroinjection of rho GDI with the guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)-bound form of rhoA p21, but not with the GTP gamma S-bound form of rhoA p21 lacking the C-terminal three amino acids, which was not post-translationally modified with lipids. The GTP gamma S-bound form of rac1 p21, the same form of G25K, the same form of smg p21B, or Ki-rasval12 p21 was ineffective. Microinjection of the bacterial ADP-ribosyltransferase C3 specific for rho p21 into Swiss 3T3 cells induced the similar changes of morphology and stress fibers. This C3 action was not prevented by comicroinjection of C3 with the GTP gamma S-bound form of rhoA p21, but was prevented by comicroinjection with the same form of a rhoA p21 mutant which was not ADP-ribosylated by C3. These results indicate that the rho GDI-rho p21 system regulates cell morphology presumably through the actomyosin system in Swiss 3T3 cells.

The molecular mechanism of the regulation of Ca2+ pump ATPase by phospholamban in cardiac sarcoplasmic reticulum was examined using synthetic peptides of phospholamban and purified Ca2+ pump ATPase from cardiac sarcoplasmic reticulum. The phospholamban monomer of 52 amino acid residues contains two distinct domains, the cytoplasmic (amino acids 1-30) and the transmembrane (amino acids 31-52) domains. The peptide corresponding to the amino acids 1-31 of phospholamban (PLN 1-31) decreased the Vmax of the Ca(2+)-dependent ATPase activity in dose-dependent manner, while it had no effect on the affinity of the ATPase for Ca2+ (KCa). On the other hand, the peptide corresponding to the amino acids 28-47 of phospholamban (PLN 28-47) increased the KCa from 0.52 to 1.33 microM without significant change in the Vmax value when reconstituted into vesicles with the ATPase. Essentially the same results as PLN 28-47 were obtained with the peptide corresponding to the amino acids 8-47 of phospholamban (PLN 8-47). The inhibitory effects of PLN 1-31 and PLN 8-47 on the ATPase were reversed by cAMP-dependent phosphorylation of the peptides (Ser16). These results indicate that phospholamban suppresses Ca2+ pump ATPase at two different sites, the cytoplasmic domain for Vmax and the transmembrane domain for KCa, and that cAMP-dependent phosphorylation de-suppresses these inhibitory effects on the ATPase.

ras p21 GTPase-activating protein (GAP) has been proposed to interact with the putative effector domain of ras p21s, and smg p21, a ras p21-like guanine nucleotide binding protein (G protein), has been shown to have the same amino acid sequence as ras p21s in this region. In the present studies, we examined the effects of ras p21 GAP on the GTPase activity of smg p21 purified from human platelets, of smg p21 on the ras p21 GAP-stimulated GTPase activity of c-Ha-ras p21 purified from Escherichia coli, and of c-Ha-ras p21 on the smg p21 GAP1- or -2-stimulated GTPase activity of smg p21. ras p21 GAP stimulated the GTPase activity of c-Ha-ras p21 but not that of smg p21. The GTP-bound form of smg p21, however, inhibited the ras p21 GAP-stimulated GTPase activity of c-Ha-ras p21 in a dose-dependent manner. The half-maximum inhibition by smg p21 was obtained at 0.4 microM which was more potent than previously observed for ras p21 (2-200 microM). The GDP-bound form also inhibited the ras p21 GAP-stimulated GTPase activity of c-Ha-ras p21, but the efficiency was 40-50% that of the GTP-bound form. smg p21 GAP1 and -2 stimulated the GTPase activity of smg p21 but not that of c-Ha-ras p21. c-Ha-ras p21 did not inhibit the smg p21 GAP1- or -2-stimulated GTPase activity of smg p21. These results indicate that ras p21 GAP interacts with smg p21 without the subsequent stimulation of its GTPase activity.