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R G Wells

Publications and source records attributed to R G Wells.

At least 19 recordsLinked to original sources
PubMed1999

Transforming growth factor-beta induces formation of a dithiothreitol-resistant type I/Type II receptor complex in live cells.

Transforming growth factor-beta (TGF-beta) binds to and signals via two serine-threonine kinase receptors, the type I (TbetaRI) and type II (TbetaRII) receptors. We have used different and complementary techniques to study the physical nature and ligand dependence of the complex formed by TbetaRI and TbetaRII. Velocity centrifugation of endogenous receptors suggests that ligand-bound TbetaRI and TbetaRII form a heteromeric complex that is most likely a heterotetramer. Antibody-mediated immunofluorescence co-patching of epitope-tagged receptors provides the first evidence in live cells that TbetaRI. TbetaRII complex formation occurs at a low but measurable degree in the absence of ligand, increasing significantly after TGF-beta binding. In addition, we demonstrate that pretreatment of cells with dithiothreitol, which inhibits the binding of TGF-beta to TbetaRI, does not prevent formation of the TbetaRI.TbetaRII complex, but increases its sensitivity to detergent and prevents TGF-beta-activated TbetaRI from phosphorylating Smad3 in vitro. This indicates that either a specific conformation of the TbetaRI. TbetaRII complex, disrupted by dithiothreitol, or direct binding of TGF-beta to TbetaRI is required for signaling.

Activin Receptors, Type I↗
PubMed1998

Oligomeric structure of type I and type II transforming growth factor beta receptors: homodimers form in the ER and persist at the plasma membrane.

Transforming growth factor beta (TGF-beta) signaling involves interactions of at least two different receptors, types I (TbetaRI) and II (TbetaRII), which form ligand-mediated heteromeric complexes. Although we have shown in the past that TbetaRII in the absence of ligand is a homodimer on the cell surface, TbetaRI has not been similarly investigated, and the site of complex formation is not known for either receptor. Several studies have indicated that homomeric interactions are involved in TGF-beta signaling and regulation, emphasizing the importance of a detailed understanding of the homooligomerization of TbetaRI or TbetaRII. Here we have combined complementary approaches to study these homomeric interactions in both naturally expressing cell lines and cells cotransfected with various combinations of epitope-tagged type I or type II receptors. We used sedimentation velocity of metabolically labeled receptors on sucrose gradients to show that both TbetaRI and TbetaRII form homodimer-sized complexes in the endoplasmic reticulum, and we used coimmunoprecipitation studies to demonstrate the existence of type I homooligomers. Using a technique based on antibody-mediated immunofluorescence copatching of receptors carrying different epitope tags, we have demonstrated ligand-independent homodimers of TbetaRI on the surface of live cells. Soluble forms of both receptors are secreted as monomers, indicating that the ectodomains are not sufficient to mediate homodimerization, although TGF-beta1 is able to promote dimerization of the type II receptor ectodomain. These findings may have important implications for the regulation of TGF-beta signaling.

Activin Receptors, Type I↗
PubMed1997

Biosynthesis of the type I and type II TGF-beta receptors. Implications for complex formation.

The TGF-beta type I and type II receptors (TbetaRI and TbetaRII) are signaling receptors that form heteromeric cell surface complexes with the TGF-betas as one of the earliest events in the cellular response to these multifunctional growth factors. Using TGF-beta-responsive mink lung epithelial cells (Mv1Lu), we have determined the half-lives of the endoplasmic reticulum (ER) and mature forms of these receptors. In metabolically labeled cells, approximately 90% of newly synthesized type II receptor undergoes modification of N-linked sugars in the Golgi, with a half-life of 30-35 min; the Golgi-processed form of the receptor has a relatively short metabolic half-life of 2.5 h. In contrast, only 50% of pulse-labeled type I receptor is converted to the Golgi-processed and therefore endoglycosidase H-resistant form, and the endoglycosidase H-sensitive ER form has a half-life of 2.8-3 h. Addition of 100 pM TGF-beta1 causes the Golgi-processed type II receptor to become less stable, with a half-life of 1.7 h, and also destabilizes the Golgi-processed type I receptor. TGF-beta1 binding and cross-linking experiments on cells treated with tunicamycin for various times confirm different ER to cell surface processing times for TbetaRI and TbetaRII. Our results, which suggest that stable complexes between type I and II TGF-beta receptors do not form until the proteins reach a post-ER compartment (presumably the cell surface), have important implications for our understanding of complex formation and receptor regulation.

Activin Receptors, Type I↗
PubMed1996

Wire stent for tracheomalacia in a five-year-old girl.

A wire stent was used successfully to treat life-threatening tracheomalacia in a 5-year-old girl. Wire stents placed bronchoscopically are nonobstructing and have the potential for balloon expansion to accommodate growth.

Bronchoscopy↗
PubMed1995

Molecular characteristics of Na(+)-coupled glucose transporters in adult and embryonic rat kidney.

Two distinct Na(+)-coupled glucose transporters (SGLTs) with either a high or a low affinity for glucose were shown to provide reabsorption of filtered glucose in the kidney. We have previously reported the characteristics of the high affinity Na+/glucose cotransporter SGLT1 from rabbit, rat, and human kidney and the low affinity Na+/glucose cotransporter SGLT2 from human kidney. Because the molecular identity of SGLT2 as the kidney cortical low affinity Na+/glucose cotransporter has been recently challenged based on studies of the porcine low affinity Na+/glucoe cotransporter SAAT-pSGLT2 (Mackenzie, B., Panayotova-Heiermann, M., Loo, D. D. F., Lever, J.E., and Wright, E. M. (1994) J. Biol. Chem. 269, 22488-22491), we have reevaluated the properties of SGLT2 in greater detail. We furthermore report new data on the regulation of SGLT1 and SGLT2 during kidney development. To analyze and compare SGLT1 and SGLT2 in adult and embryonic kidney, we have cloned and characterized SGLT2 from rat kidney and determined its tissue distribution based on Northern analysis and in situ hybridization. When expressed in Xenopus oocytes, rat SGLT2 stimulated transport of alpha-methyl-D-glucopyranoside (2 mM) in oocytes up to 4.5-fold over controls with an apparent Km of 3.0 mM. The transport properties (i.e. a Na+ to glucose coupling of 1:1 and lack of galactose transport) generally matched those of the kidney cortical low affinity system. We show that expression of rat SGLT2 mRNA is kidney specific and that it is strongly and exclusively expressed in proximal tubule S1 segments. Hybrid-depletion studies were performed to conclusively determine whether SGLT2 corresponds to the kidney cortical low affinity system. Injection of rat kidney superficial cortex mRNA into oocytes stimulated the uptake of alpha-methyl-D-glucopyranoside (2 mM) 2-3-fold. We show that hybrid depletion of this kidney RNA using an SGLT2 antisense oligonucleotide completely suppresses the uptake. These data strongly indicate that SGLT2 is the major kidney cortical low affinity glucose transporter. We therefore propose that SAAT-pSGLT2 be renamed SGLT3. Experiments addressing the expression of SGLT1 and SGLT2 mRNAs in embryonic rat kidneys reveal that the two Na+/glucose cotransporters are developmentally regulated and that there may be a different splice variant for SGLT2 in embryonic kidney compared to the adult.

Age Factors↗
PubMed1995

The soluble exoplasmic domain of the type II transforming growth factor (TGF)-beta receptor. A heterogeneously glycosylated protein with high affinity and selectivity for TGF-beta ligands.

The transforming growth factor (TGF)-beta type II receptor is a transmembrane serine/threonine kinase which is essential for all TGF-beta-induced signals. In several cell types TGF-beta 2 is as potent as TGF-beta or TGF-beta 3 in inducing cellular responses, yet TGF-beta 2 does not bind to the majority of expressed type II receptors. Here we characterized the properties of the soluble extracellular domain of the human TGF-beta type II receptor synthesized in COS-7 cells. Like the membrane-attached type II receptor, the soluble receptor contains complex N-linked oligosaccharides as well as additional sialic acid residues that cause it to migrate heterogenously upon SDS-polyacrylamide gel electrophoresis. 125I-TGF-beta 1 binds to and is chemically cross-linked to this protein. Unlabeled TGF-beta 1 inhibits the binding of 125I-TGF-beta 1 with an apparent dissociation constant (Kd) of approximately 200 pM, similar to the apparent Kd (approximately 50 pM) of the cell-surface type II receptor. TGF-beta 3 inhibits the binding of 125I-TGF-beta 1 to the soluble type II receptor with a similar dissociation constant, approximately 500 pM. In contrast, 125I-TGF-beta 2 cannot bind and be chemically cross-linked to the soluble type II receptor, nor does as much as a 125-fold excess of unlabeled TGF-beta 2 inhibit the binding of 125I-TGF-beta 1 to the soluble receptor. This is the first demonstration of the binding affinities of the type II receptor in the absence of the other cell-surface molecules known to bind TGF-beta. Expressed alone in COS-7 cells the type II receptor also cannot bind TGF-beta 2; co-expression of type III receptor enables the type II receptor to bind TGF-beta 2. Thus, the type III receptor or some other component is required for transmission of TGF-beta 2-induced signals by the type II receptor.

Animals↗
PubMed1994

The high affinity Na+/glucose cotransporter. Re-evaluation of function and distribution of expression.

We report the primary structure, functional characterization, and tissue distribution of the high affinity Na+/glucose cotransporter SGLT1 from rat kidney. Rat SGLT1 (665 amino acid residues) is 86-87% identical to SGLT1 from rabbit, pig, and human. High stringency Northern analysis demonstrated that SGLT1 is strongly expressed in small intestine and at lower levels in kidney, liver, and lung. In situ hybridization performed on kidney sections revealed that SGLT1 is predominantly present in S3 segments of the proximal tubule. In small intestine, SGLT1 message was located in cells of the lower two-thirds of intestinal villi. Expression of rat SGLT1 in Xenopus oocytes resulted in a large Na(+)-dependent uptake of [14C]-alpha-methyl-D-glucopyranoside (alpha MeGlc). Overall, the transport characteristics were similar to those of rabbit SGLT1. High affinity Na+/glucose cotransport in membrane vesicles was previously shown to be coupled to the cotransport of two Na+ ions (Turner, R. J., and Moran, A. (1982) J. Membr. Biol. 70, 37-45). Previous kinetic analysis of rat and rabbit SGLT1, however, demonstrated between second and first order dependence of sugar uptake on extracellular Na+ concentration, suggesting the existence of Na(+)-binding sites with different affinities. Here, we directly compared the initial rates of the alpha MeGlc uptake with alpha MeGlc-induced inward currents as an indicator of the Na+ flux. This analysis clearly revealed a Na+ to glucose coupling ratio of 2:1. In summary, our data provide important insights into the function and tissue distribution of the high affinity Na+/glucose cotransporter SGLT1 and clarify its role in the reabsorption mechanism of D-glucose in the kidney.

Amino Acid Sequence↗
PubMed1994

AIDS: sonography of hyperechoic renal pyramids in renal candidiasis.

A 13-month-old patient with autoimmune deficiency syndrome had renal candidiasis. An abnormal renal sonographic pattern of hyperechoic renal pyramids was demonstrated. This abnormal sonographic finding should suggest renal candidiasis in an immune suppressed child. In the normal kidney of children, the pyramids are hypoechoic. This pattern has not been noted in previous publications. We report this case.

AIDS-Associated Nephropathy↗
PubMed1994

Adrenal hyperechogenicity in hemolytic uremic syndrome.

Hemolytic uremic syndrome is characterized by an abrupt onset of a microangiopathy, thrombocytopenia, hemolytic anemia and renal failure. These abnormalities present in varying degrees. The vascular abnormalities include endothelial swelling and thrombus formation. The kidney is the major target of involvement, although intestines, lung and brain may also be affected. It is considered a disease of the infant, although it occurs in older children and adults as a component of thrombotic thrombocytopenic purpura. We present the sonographic findings of adrenal involvement in a child with hemolytic uremic syndrome, which seems to be a rare occurrence. The patient developed adrenal insufficiency during the acute phase of the disease.

Adrenal Glands↗