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Biomedical subjects

Yashoda Ghanekar

Publications and source records attributed to Yashoda Ghanekar.

5 recordsLinked to original sources
PubMed2005

Protein kinase D: activation for Golgi carrier formation.

Protein kinase D regulates fission at the trans-Golgi network (TGN) of transport carriers that deliver cargo to the plasma membrane. PKD is first recruited to the TGN through interaction with diacylglycerol and is subsequently activated by phosphorylation to promote carrier fission. In a recent study, the relevant upstream kinase at the TGN was identified as the novel protein kinase C isoform PKCeta, which in turn is activated in response to heterotrimeric G-protein activation. These findings indicate the existence of a kinase signaling cascade at the TGN that regulates carrier fission and suggest a mechanism by which cargo might direct the formation of its transport carriers.

Animals↗
PubMed2004

Glycosylation of the receptor guanylate cyclase C: role in ligand binding and catalytic activity.

GC-C (guanylate cyclase C) is the receptor for heat-stable enterotoxins, guanylin and uroguanylin peptides. Ligand binding to the extracellular domain of GC-C activates the guanylate cyclase domain leading to accumulation of cGMP. GC-C is expressed as differentially glycosylated forms in HEK-293 cells (human embryonic kidney-293 cells). In the present study, we show that the 145 kDa form of GC-C contains sialic acid and galactose residues and is present on the PM (plasma membrane) of cells, whereas the 130 kDa form is a high mannose form that is resident in the endoplasmic reticulum and serves as the precursor for the PM-associated form. Ligand-binding affinities of the differentially glycosylated forms are similar, indicating that glycosylation of GC-C does not play a role in direct ligand interaction. However, ligand-stimulated guanylate cyclase activity was observed only for the fully mature form of the receptor present on the PM, suggesting that glycosylation had a role to play in imparting a conformation to the receptor that allows ligand stimulation. Treatment of cells at 20 degrees C led to intracellular accumulation of a mature glycosylated form of GC-C that now showed ligand-stimulated guanylate cyclase activity, indicating that localization of GC-C was not critical for its catalytic activity. To determine if complex glycosylation was required for ligand-stimulated activation of GC-C, the receptor was expressed in HEK-293 cells that were deficient in N -acetylglucosaminyltransferase 1. This minimally glycosylated form of the receptor was expressed on the cell surface and could bind a ligand with an affinity comparable with the 145 kDa form of the receptor. However, this form of the receptor was poorly activated by the ligand. Therefore our studies indicate a novel role for glycosidic modification of GC-C during its biosynthesis, in imparting subtle conformational changes in the receptor that allow for ligand-mediated activation and perhaps regulation of basal activity.

Amino Acid Sequence↗
PubMed2003

Cellular refractoriness to the heat-stable enterotoxin peptide is associated with alterations in levels of the differentially glycosylated forms of guanylyl cyclase C.

The heat-stable enterotoxin peptides (ST) produced by enterotoxigenic Escherichia coli are one of the major causes of transitory diarrhea in the developing world. Toxin binding to its receptor, guanylyl cyclase C (GC-C), results in receptor activation and the production of high intracellular levels of cGMP. GC-C is expressed in two differentially glycosylated forms in intestinal epithelial cells. Prolonged exposure of human colonic cell lines to ST peptides induces cellular refractoriness to the ST peptide, in terms of intracellular cGMP accumulation. We have investigated the mechanism of cellular desensitization in human colonic Caco2 cells, and observe that exposure of cells to ST leads to a time and dose-dependent inability of cells to respond to the peptide in terms of GC-C stimulation, both in whole cells and membranes prepared from desensitized cells. This is concomitant with a 50% reduction in ST-binding activity in desensitized cells. Desensitization was correlated with a loss of the plasma membrane-associated, hyperglycosylated 145 kDa form of GC-C, while the predominant 130 kDa form, localized both on the plasma membrane and the endoplasmic reticulum, continued to be present in ST-treated cells. Desensitized cells recovered ST-responsiveness on removal of the ST peptide, which was correlated with a reappearance of the 145 kDa form on the cell surface, following processing of the endoplasmic reticulum-associated pool of the 130 kDa form. Selective internalization of the 145 kDa form of the receptor was required for cellular desensitization, as ST-treatment of cells at 4 degrees C did not lead to refractoriness. We therefore show a novel means of regulation of cellular responsiveness to the ST peptide, whereby altering cellular levels of the differentially glycosylated forms of GC-C can lead to differential ligand-mediated activation of the receptor.

Bacterial Toxins↗
PubMed2002

Guanylin, uroguanylin, and heat-stable euterotoxin activate guanylate cyclase C and/or a pertussis toxin-sensitive G protein in human proximal tubule cells.

Membrane guanylate cyclase C (GC-C) is the receptor for guanylin, uroguanylin, and heat-stable enterotoxin (STa) in the intestine. GC-C-deficient mice show resistance to STa in intestine but saluretic and diuretic effects of uroguanylin and STa are not disturbed. Here we describe the cellular effects of these peptides using immortalized human kidney epithelial (IHKE-1) cells with properties of the proximal tubule, analyzed with the slow-whole-cell patch clamp technique. Uroguanylin (10 or 100 nm) either hyperpolarized or depolarized membrane voltages (V(m)). Guanylin and STa (both 10 or 100 nm), as well as 8-Br-cGMP (100 microm), depolarized V(m). All peptide effects were absent in the presence of 1 mm Ba(2+). Uroguanylin and guanylin changed V(m) pH dependently. Pertussis toxin (1 microg/ml, 24 h) inhibited hyperpolarizations caused by uroguanylin. Depolarizations caused by guanylin and uroguanylin were blocked by the tyrosine kinase inhibitor, genistein (10 microm). All three peptides increased cellular cGMP. mRNA for GC-C was detected in IHKE-1 cells and in isolated human proximal tubules. In IHKE-1 cells GC-C was also detected by immunostaining. These findings suggest that GC-C is probably the receptor for guanylin and STa. For uroguanylin two distinct signaling pathways exist in IHKE-1 cells, one involves GC-C and cGMP as second messenger, the other is cGMP-independent and connected to a pertussis toxin-sensitive G protein.

Bacterial Toxins↗