Secretion and mesoderm-inducing activity of the TGF-beta-related domain of Xenopus Vg1

Vg1 is a maternal mRNA localized to the vegetal hemisphere of Xenopus embryos during blastula stages, a region responsible for the induction of mesoderm in the adjacent marginal zone. Its homology to the transforming growth factor-beta family, which includes several proteins with mesoderm-inducing activity, suggests a role for Vg1 as an endogenous mesoderm-inducing factor. However, expression of Vg1 protein in the animal hemisphere, following injection of synthetic mRNA, has no effect on development, and isolated animal caps are not mesodermalized. It is shown that Vg1 protein fails to form dimers and is not processed to release the putative bioactive domain. Furthermore it is shown that the N-terminal signal peptide of Vg1 is not cleaved following translocation into the ER, which may explain the failure of this protein to dimerize. To explore the role of Vg1 in amphibian development, a fusion protein has been made of the preproregion of Xenopus bone morphogenetic protein-4 and the putative bioactive C-terminal domain of Vg1. This fusion protein forms dimers and the C-terminal domain of Vg1 is secreted. Injection of this construct into Xenopus embryos induces the formation of a second dorsal axis and isolated animal caps are mesodermalized. The results are consistent with a role for Vg1 in mesoderm induction during Xenopus development.     

Mutant Vg1 ligands disrupt endoderm and mesoderm formation in Xenopus embryos

The Xenopus Vg1 gene, a TGFbeta superfamily member, is expressed as a maternal mRNA localized to prospective endoderm, and mature Vg1 protein can induce both endodermal and mesodermal markers in embryonic cells. Most previous work on embryonic inducers, including activin, BMPs and Vg1, has relied on ectopic expression to assay for gene function. Here we employ a mutant ligand approach to block Vg1 signaling in developing embryos. The results indicate that Vg1 expression is essential for normal endodermal development and the induction of dorsal mesoderm in vivo.     

Patterns of localization and cytoskeletal association of two vegetally localized RNAs, Vg1 and Xcat-2

In Xenopus, localization of a rare class of mRNAs during oogenesis is believed to initiate pattern formation in the early embryo. We have determined the pattern of RNA localization for one of these RNAs, Xcat-2, which encodes a putative RNA-binding protein related to Drosophila nanos (Mosquera, L., Forristall, C., Zhou, Y. and King, M. L. (1993) Development 117, 377-386). Xcat-2 is exclusively localized to the mitochondrial cloud in stage I oocytes, moves with this body into the vegetal cortex during stage II and, later, partitions into islands consistent with it being a component of the germ plasm. As previously shown, Vg1 is not localized to the vegetal cortex until stage IV and distributes to all vegetal blastomeres during development. We found a direct correlation between the localized condition of these RNAs and their recovery in a detergent-insoluble fraction. We present evidence suggesting that differential RNA binding to a cytoskeletal component(s) in the vegetal cortex determines the pattern of inheritance for that RNA in the embryo.     

Blocking effects of benzyltetrahydropalmatine on delayed rectified K+ currents expressed in Xenopus oocytes and in toad oocytes

The effects of benzyltetrahydropalmatine (BTHP) on delayed rectified K+ currents (Ik) expressed in Xenopus oocytes and Ik of toad (Bufo bufo gargarizans) oocytes were studied. The Ik expressed in Xenopus oocytes was measured after microinjection of mRNA isolated from carp fish (C anratus L.) brains with double -microelectrode voltage clamp technique. The maximum and mean value of Ik expressed in Xenopus oocytes were 600 nA and 360 +/- 104 nA, respectively. BTHP reduced the current amplitude of Ik expressed in Xenopus oocytes in 10-1000 mumol.L-1 dose-dependently, EC50 was 29 mumol.L-1. Also, the reduction of Ik of toad oocytes was 9.1%, 29.1%, 54.7% and 68.6% by BTHP 10, 30, 100 and 1000 mumol.L-1, respectively, EC50 was 33 mumol.L-1. The results showed that BTHP possesses an inhibitory effect on Ik, the main ion mechanism of antiarrhythmic action of BTHP.     

Induction of axial mesoderm by zDVR-1, the zebrafish orthologue of Xenopus Vg1

The zebrafish DVR-1 (zDVR-1) gene, like Xenopus Vg1, is present maternally as an unprocessed precursor protein which is distributed ubiquitously along the future dorsoventral axis. Also, like Vg1, overexpression of zDVR-1 in zebrafish directs synthesis of more precursor, but no processed protein. However, the native zDVR-1 precursor is processed to mature protein when expressed in Xenopus. Like processed Vg1, mature zDVR-1 is a potent inducer of axial mesoderm. The parallels in expression pattern, apparent regulation of protein processing, and mesoderm-inducing activity support the hypothesis that localized protein processing controls production of a dorsal mesoderm inducer in these two species. Furthermore, using mutant mRNAs, we show that cleavage site sequences of the precursor protein are important in regulating protein processing.     

Ribonucleoprotein formation by the ORF1 protein of the non-LTR retrotransposon Tx1L in Xenopus oocytes

The Tx1L elements constitute a family of site-specific non-LTR retrotransposons found in the genome of the frog Xenopus laevis . The elements have two open reading frames (ORFs) with homology to proteins of retroviruses and other retroelements. This study demonstrates an expected activity of one of the element-encoded proteins. The RNA binding properties of ORF1p, the product of the first ORF of Tx1L, were examined after expression from RNA injected into Xenopus oocytes. Using sucrose gradient sedimentation and non-denaturing gel electrophoresis, we show that ORF1p associates with RNA in cytoplasmic ribonucleoprotein (RNP) particles. Discrete RNPs are formed with well-defined mobilities. The ORF1p RNPs are distinct from endogenous RNPs that contain stored oocyte mRNAs and two specific endogenous mRNAs do not become associated with ORF1p. ORF1p appears to be capable of associating with its own mRNA and with other injected RNAs, independent of specific recognition sequences. Although nuclear localization of ORF1p was anticipated, based both on the supposed mechanism of transposition and on the presence of a potential nuclear localization signal, no significant fraction of the protein was found in the oocyte nucleus. Nonetheless, the RNA binding capability of ORF1p is consistent with the proposed model for transposition of non-LTR retrotransposons.     

Native serotonin 5-HT3 receptors expressed in Xenopus oocytes differ from homopentameric 5-HT3 receptors

Efficacies of the 5-hydroxytryptamine (serotonin) 5-HT3 receptor (5-HT3R) agonists 2-methyl-5-HT, dopamine, and m-chlorophenylbiguanide on 5-HT3R native to N1E-115 cells and on homopentameric 5-HT3R expressed in Xenopus oocytes were determined relative to that of 5-HT. Efficacies of 2-methyl-5-HT and dopamine on 5-HT3R native to differentiated N1E-115 cells are high (54 and 36%) as compared with their efficacies on homopentameric 5-HT3R-A(L) and 5-HT3R-A(S) receptors expressed in oocytes (4-8%). m-Chlorophenylbiguanide does not distinguish between 5-HT3R in N1E-115 cells and in oocytes. The distinct pharmacological profile of 5-HT3R native to differentiated N1E-115 cells is conserved when poly(A)+ mRNA from these cells is expressed in oocytes. The results indicate that, apart from the known 5-HT3R subunits, N1E-115 cells express additional proteins involved in 5-HT3R function.     

Expression of rat liver cell membrane transporters for thyroid hormone in Xenopus laevis oocytes

The present study was conducted to explore the possible use of Xenopus laevis oocytes for the expression cloning of cell membrane transporters for iodothyronines. Injection of stage V-VI X. laevis oocytes with 23 ng Wistar rat liver polyadenylated RNA (mRNA) resulted after 3-4 days in a highly significant increase in [125I]T3 (5 nM) uptake from 6.4 +/- 0.8 fmol/oocyte x h in water-injected oocytes to 9.2 +/- 0.65 fmol/oocyte x h (mean +/- SEM; n = 19). In contrast, [125I]T4 (4 nM) uptake was not significantly stimulated by injection of total liver mRNA. T3 uptake induced by liver mRNA was significantly inhibited by replacement of Na+ in the incubation medium by choline+ or by simultaneous incubation with 1 microM unlabeled T3. In contrast, T3 uptake by water-injected oocytes was not Na+ dependent. Fractionation of liver mRNA on a 6-20% sucrose gradient showed that maximal stimulation of T3 uptake was obtained with mRNA of 0.8-2.1 kilobases (kb). In contrast to unfractionated mRNA, the 0.7- to 2.1-kb fraction also significantly stimulated transport of T4, and it was found to induce uptake of T3 sulfate (T3S). Because T3S is a good substrate for type I deiodinase (D1), 2.3 ng rat D1 complementary RNA (cRNA) were injected either alone or together with 23 ng of the 0.8- to 2.1-kb fraction of rat liver mRNA. Compared with water-injected oocytes, injection of D1 cRNA alone did not stimulate uptake of [125I]T3S (1.25 nM). T3S uptake in liver mRNA and D1 cRNA-injected oocytes was similar to that in oocytes injected with mRNA alone, showing that transport of T3S is independent of the metabolic capacity of the oocyte. Furthermore, coinjection of liver mRNA and D1 cRNA strongly increased the production of 125I-, showing that the T3S taken up by the oocyte is indeed transported to the cell interior. In conclusion, injection of rat liver mRNA into X. laevis oocytes resulted in a stimulation of saturable, Na+-dependent T4, T3 and T3S transport, indicating that rat liver contains mRNA(s) coding for plasma membrane transporters for these iodothyronine derivatives.     

Expression of a human renal sodium nucleoside cotransporter in Xenopus laevis oocytes

In this study, Xenopus laevis oocytes injected with poly(A)+ RNA (mRNA) isolated from human kidney were used to express a Na(+)-nucleoside cotransporter. Na(+)-stimulated [3H]thymidine uptake was enhanced 2-3-fold in oocytes injected with 50 ng poly(A)+ RNA and 4-5-fold in oocytes injected with 20 ng of a size-fractionated human renal cortex mRNA fragment (2-3 kb) in comparison with water-injected oocytes. Na(+)-dependent thymidine uptake in oocytes injected with the 2-3 kb mRNA fragment was inhibited significantly by thymidine and guanosine but not by formycin B, consistent with the N4 Na(+)-nucleoside cotransporter. The Km (28 microM) of Na(+)-dependent thymidine uptake in the oocytes injected with the 2-3 kb mRNA fragment was similar to the Km (27 microM) of Na(+)-dependent thymidine uptake obtained in human renal brush border membrane vesicles. These data suggest for the first time that a Na(+)-nucleoside cotransporter from human kidney can be expressed in X. laevis oocytes.     

Cloning of a Xenopus laevis inwardly rectifying K+ channel subunit that permits GIRK1 expression of IKACh currents in oocytes

Xenopus oocytes injected with GIRK1 mRNA express inwardly rectifying K+ channels resembling IKACh. Yet IKACh, the atrial G protein-regulated ion channel, is a heteromultimer of GIRK1 and CIR. Reasoning that an oocyte protein might be substituting for CIR, we cloned XIR, a CIR homolog endogenously expressed by Xenopus oocytes. Coinjecting XIR and GIRK1 mRNAs produced large, inwardly rectifying K+ currents responsive to m2-muscarinic receptor stimulation. The m2-stimulated currents of oocytes expressing GIRK1 alone decreased 80% after injecting antisense oligonucleotides specific to the 5' untranslated region of XIR, but GIRK1/CIR currents were unaffected. Thus, GIRK1 without XIR or CIR only ineffectively produces currents in oocytes. This result suggests that GIRK1 does not form native homomultimeric channels.     

A rho-associated protein kinase, ROKalpha, binds insulin receptor substrate-1 and modulates insulin signaling

Insulin receptor substrate-1 (IRS-1) is phosphorylated on multiple tyrosine residues by ligand-activated insulin receptors. These tyrosine phosphorylation sites serve to dock several Src homology 2-containing signaling proteins. In addition, IRS-1 contains a pleckstrin homology domain and a phosphotyrosine binding domain (PTB) implicated in protein-protein and protein-lipid interactions. In a yeast two-hybrid screening using Xenopus IRS-1 (xIRS-1) pleckstrin homology-PTB domains as bait, we identified a Xenopus homolog of Rho-associated kinase alpha (xROKalpha) as a potential xIRS-1-binding protein. The original clone contained the carboxyl terminus of xROKalpha (xROK-C) including the putative Rho binding domain but lacking the amino-terminal kinase domain. Further analyses in yeast indicated that xROK-C bound to the putative PTB domain of xIRS-1. Binding of xROK-C to xIRS-1 was confirmed in Xenopus oocytes after microinjection of mRNA corresponding to xROK-C. Furthermore, microinjection of xROK-C mRNA inhibited insulin-induced mitogen-activated protein kinase activation with a concomitant inhibition of oocyte maturation. In contrast, microinjection of xROK-C mRNA did not inhibit mitogen-activated protein kinase activation or oocyte maturation induced by progesterone or by microinjection of viral Ras (v-Ras) mRNA. These results suggest that xROKalpha may play a role in insulin signaling via a direct interaction with xIRS-1.     

TGF-beta inhibits IL-2-induced tyrosine phosphorylation and activation of Jak-1 and Stat 5 in T lymphocytes

The major yolk protein precursor in mosquito oocytes, vitellogenin (Vg), is internalized by a 205-kDa membrane-bound receptor (VgR). Recently, VgR has been isolated permitting the production of polyclonal anti-VgR antibodies. To elucidate the pathway of VgR internalization and recycling in mosquito oocytes during Vg uptake, we carried out an immunogold electron-microscopic study, labeling both Vg and VgR in ultrathin frozen sections of ovarian tissue. VgR immunolabeling demonstrated that the oocyte plasma membrane was subdivided into microdomains, with VgR being located between and at the lower portions of the oocyte microvilli. During the early stages of internalization, Vg and VgR were observed together in coated pits, coated vesicles, and early endosomes. Fusion of early endosomes created transitional yolk bodies (TYB) in which Vg and VgR became segregated. VgR label was present in the numerous tubular compartments that protruded from the TYBs. These tubular organelles extended to and fused with the plasma membrane, suggesting that they represented the vehicle for VgR recycling. Vg label was not observed in the tubular compartments. Instead, Vg accumulated in the core of the TYB, a region free of VgR label. Mature yolk bodies (MYB) were heavily labeled for Vg, but completely lacked any VgR label, indicating that MYB are storage compartments that do not participate in receptor recycling. Thus, our immunocytochemical data clearly visualize the steps in Vg/VgR internalization, dissociation, sorting, and recycling of the receptor to the plasma membrane.     

Hepatic sinusoidal membrane transport of anionic drugs mediated by anion transporter Npt1

The purpose of our study was to establish the localization of the anion transporter Npt1 in liver and the relevance of Npt1 to carrier-mediated hepatic transport of beta-lactam antibiotics. Immunocytochemical examination of mouse liver with antiserum for Npt1 showed basolateral (sinusoidal) membrane localization. Function of Npt1 was characterized in Xenopus laevis oocytes. Injection of in vitro-transcribed cRNA into oocytes resulted in an increased uptake of [14C]benzylpenicillin (PCG). The Npt1-mediated uptake was saturable with a Michaelis constant (Km) of 0.46 +/- 0.18 mM and a maximum rate (Vmax) of 46.6 +/- 8.5 pmol/60 min/oocyte, and the uptake of [14C]PCG was independent of Na+ and pH, but dependent on chloride ion. Npt1-mediated [14C]PCG uptake was inhibited by several beta-lactam antibiotics and probenecid. Oocytes injected with Npt1-cRNA demonstrated significantly enhanced transport activity for other anionic compounds such as [14C]faropenem, [14C]foscarnet and [3H]mevalonic acid, as well as [14C]PCG, compared with water-injected oocytes. In conclusion, Npt1 is suggested to participate in hepatic sinusoidal membrane transport of organic anions such as beta-lactam antibiotics as well as inorganic anions for the efflux from hepatocyte-to-blood direction.     

A novel insulin receptor substrate protein, xIRS-u, potentiates insulin signaling: functional importance of its pleckstrin homology domain

A novel Xenopus insulin receptor substrate cDNA was isolated by hybridization screening using the rat insulin receptor substrate-1 (IRS-1) cDNA as a probe. The xIRS-u cDNA encodes an open reading frame of 1003 amino acids including a putative amino-terminal pleckstrin homology (PH) domain and phosphotyrosine-binding (PTB) domain. The carboxy terminus of xIRS-u contains several potential Src homology 2 (SH2)-binding sites, five of which are in the context of YM/LXM (presumptive binding sites for phosphatidylinositol 3-kinase). It also contains a putative binding site for Grb2 (YINID). Pair-wise amino acid sequence comparisons with the previously identified xIRS-1 and the four members of the mammalian IRS family (1 through 4) indicated that xIRS-u has similar overall sequence homology (33-45% identity) to all mammalian IRS proteins. In contrast, the previously isolated xIRS-1 is particularly similar (67% identical) to IRS-1 and considerably less similar (31-46%) to the other IRS family members (2 through 4). xIRS-u is also distinct from xIRS-1, having an overall sequence identity of 47%. These sequence analyses suggest that xIRS-u is a novel member of the IRS family rather than a Xenopus homolog of an existing member. Microinjection of mRNA encoding a Myc-tagged xIRS-u into Xenopus oocytes resulted in the expression of a 120-kDa protein (including 5 copies of the 13-amino acid Myc tag). The injection of xIRS-u mRNA accelerated insulin-induced MAP kinase activation with a concomitant acceleration of insulin-induced oocyte maturation. An aminoterminal deletion of the PH domain (xIRS-u deltaPH) significantly reduced the ability of xIRS-u to potentiate insulin signaling. In contrast to the full-length protein, injection of xIRS-u (1-299), which encoded the PH and PTB domain, or xIRS-u (1-170), which encoded only the PH domain, blocked insulin signaling in Xenopus oocytes. Finally, xIRS-u (119-299), which had a truncated PH domain and an intact PTB domain, had no effect on insulin signaling. This is the first report that the PH domain of an IRS protein can function in a dominant negative manner to inhibit insulin signaling.     

Germinal vesicle material is dispensable for oscillations in cdc2 and MAP kinase activities, cyclin B degradation and synthesis during meiosis in Xenopus oocytes

We have investigated at a molecular level the requirements for germinal vesicle (nuclear) material during the course of meiosis in Xenopus oocytes. We present the localization of some cell cycle proteins in stage VI oocytes; most of those analyzed are cytoplasmic, although some (MAD, 26S proteasome) are distributed between the cytoplasm and the germinal vesicle. By analyzing changes in individual oocytes, we find that the unphosphorylated form of cyclin B2 disappears and the phosphorylated form is then degraded in both nucleated and enucleated oocytes. Enucleated oocytes are also capable of resynthesizing both cyclin B1 and cyclin B2 after the initial degradation and of reactivating cdc2 kinase. Synthesis of mos protein and activation of MAP kinase concomitant with cdc2-cyclin B reactivation are also unaffected by prior removal of the germinal vesicle.     

mRNA from frog corneal epithelium increases water permeability in Xenopus oocytes

PURPOSE: To investigate the existence of a water channel in the frog corneal epithelium by studying the osmotic water permeability (Pf) of Xenopus oocytes expressing the mRNA message from frog corneal epithelium. METHODS: Total RNA was obtained from corneal epithelium by a single-step phase separation method, and poly A+ RNA was isolated using oligo-dT columns. This mRNA was injected into the oocytes. After a 48-hour incubation, oocyte volume changes elicited by a hypoosmotic solution were measured with a computerized video system. RESULTS: Oocytes injected with 50 nl mRNA (1 microgram/microliter) showed a significant increase in Pf compared to water-injected controls (8.4 +/- 1.5 to 17.5 +/- 1.9 cm.sec-1 x 10(-4), P < 0.005). mRNA-injected oocytes exposed to a higher external [Cl-] showed a heightened permeability. Furthermore, Pf of oocytes exposed to a solution containing the recognized water-channel blocker HgCl2 was significantly lower than the Pf of mRNA-injected oocytes not exposed to HgCl2. CONCLUSIONS: Evidence was found for a water channel in the frog corneal epithelium because oocytes injected with the epithelial mRNA manifested increased water permeability. The increase in water permeability was larger in the presence of external Cl- and was inhibited by HgCl2. This finding correlates with measurements of Pf in the intact epithelium in which apical Cl- induced an increase in transepithelial water permeability prevented by HgCl2.