AMP-activated protein kinase (AMPK) activation and glycogen synthase kinase-3ß (GSK-3ß) inhibition induce Ca2+-independent deposition of tight junction components at the plasma membrane.

Extracellular Ca(2+) is essential for the development of stable epithelial tight junctions. We find that in the absence of extracellular Ca(2+), AMP-activated protein kinase (AMPK) activation and glycogen synthase kinase (GSK)-3ß inhibition independently induce the localization of epithelial tight junction components to the plasma membrane. The Ca(2+)-independent deposition of junctional proteins induced by AMPK activation and GSK-3ß inhibition is independent of E-cadherin. Furthermore, the nectin-afadin system is required for the deposition of tight junction components induced by AMPK activation, but it is not required for that induced by GSK-3ß inhibition. Phosphorylation studies demonstrate that afadin is a substrate for AMPK. These data demonstrate that two kinases involved in regulating cell growth and metabolism act through distinct pathways to influence the deposition of the components of epithelial tight junctions.

A PDZ-binding motif controls basolateral targeting of syndecan-1 along the biosynthetic pathway in polarized epithelial cells.

The cell surface proteoglycan, syndecan-1, is essential for normal epithelial morphology and function. Syndecan-1 is selectively localized to the basolateral domain of polarized epithelial cells and interacts with cytosolic PDZ (PSD-95, discs large, ZO-1) domain-containing proteins. Here, we show that the polarity of syndecan-1 is determined by its type II PDZ-binding motif. Mutations within the PDZ-binding motif lead to the mislocalization of syndecan-1 to the apical surface. In contrast to previous examples, however, PDZ-binding motif-dependent polarity is not determined by retention at the basolateral surface but rather by polarized sorting prior to syndecan-1's arrival at the plasma membrane. Although none of the four known PDZ-binding partners of syndecan-1 appears to control basolateral localization, our results show that the PDZ-binding motif of syndecan-1 is decoded along the biosynthetic pathway establishing a potential role for PDZ-mediated interactions in polarized sorting.

Transcytosis of NgCAM in epithelial cells reflects differential signal recognition on the endocytic and secretory pathways.

NgCAM is a cell adhesion molecule that is largely axonal in neurons and apical in epithelia. In Madin-Darby canine kidney cells, NgCAM is targeted to the apical surface by transcytosis, being first inserted into the basolateral domain from which it is internalized and transported to the apical domain. Initial basolateral transport is mediated by a sequence motif (Y(33)RSL) decoded by the AP-1B clathrin adaptor complex. This motif is a substrate in vitro for tyrosine phosphorylation by p60src, a modification that disrupts NgCAM's ability to interact with clathrin adaptors. Based on the behavior of various NgCAM mutants, it appears that after arrival at the basolateral surface, the AP-1B interaction site is silenced by phosphorylation of Tyr(33). This slows endocytosis and inhibits basolateral recycling from endosomes, resulting in NgCAM transcytosis due to a cryptic apical targeting signal in its extracellular domain. Thus, transcytosis of NgCAM and perhaps other membrane proteins may reflect the spatial regulation of recognition by adaptors such as AP-1B.

Metabolism of phytoestrogen conjugates.

Phytoestrogens are plant-derived compounds with physiologic estrogenic effects. They are present in the plant as glycosidic conjugates, some of which contain further chemical modifications (acetate, malonate, and 3-hydroxy-3-methylglutarate esters and 2,3-dihydroxysuccinate ether). In the gastrointestinal tract, the conjugates undergo hydrolysis catalyzed by enzymes in the intestinal wall and by gut bacteria. On entering the systemic circulation, the phytoestrogens may undergo extensive metabolism to other compounds through reactions involving demethylation, methylation, hydroxylation, chlorination, iodination, and nitration. In addition, all these compounds can undergo conjugation to form beta-glucuronides and sulfate esters. This chapter describes the methods of analysis of all these compounds, the sources of or methods to manufacture suitable standards, and the procedures for examining the enzymes that catalyze these reactions.

A hierarchy of signals regulates entry of membrane proteins into the ciliary membrane domain in epithelial cells.

The membrane of the primary cilium is continuous with the plasma membrane but compositionally distinct. Although some membrane proteins concentrate in the cilium, others such as podocalyxin/gp135 are excluded. We found that exclusion reflects a saturable selective retention mechanism. Podocalyxin is immobilized by its PDZ interaction motif binding to NHERF1 and thereby to the apical actin network via ERM family members. The retention signal was dominant, autonomous, and transferable to membrane proteins not normally excluded from the cilium. The NHERF1-binding domains of cystic fibrosis transmembrane conductance regulator and Csk-binding protein were also found to act as transferable retention signals. Addition of a retention signal could inhibit the ciliary localization of proteins (e.g., Smoothened) containing signals that normally facilitate concentration in the ciliary membrane. Proteins without a retention signal (e.g., green fluorescent protein-glycosylphosphatidylinositol) were found in the cilium, suggesting entry was not impeded by a diffusion barrier or lipid microdomain. Thus, a hierarchy of interactions controls the composition of the ciliary membrane, including selective retention, selective inclusion, and passive diffusion.

Par3 functions in the biogenesis of the primary cilium in polarized epithelial cells.

Par3 is a PDZ protein important for the formation of junctional complexes in epithelial cells. We have identified an additional role for Par3 in membrane biogenesis. Although Par3 was not required for maintaining polarized apical or basolateral membrane domains, at the apical surface, Par3 was absolutely essential for the growth and elongation of the primary cilium. The activity reflected its ability to interact with kinesin-2, the microtubule motor responsible for anterograde transport of intraflagellar transport particles to the tip of the growing cilium. The Par3 binding partners Par6 and atypical protein kinase C interacted with the ciliary membrane component Crumbs3 and we show that the PDZ binding motif of Crumbs3 was necessary for its targeting to the ciliary membrane. Thus, the Par complex likely serves as an adaptor that couples the vectorial movement of at least a subset of membrane proteins to microtubule-dependent transport during ciliogenesis.

Differential biliary excretion of genistein metabolites following intraduodenal and intravenous infusion of genistin in female rats.

The purpose of this study was to determine whether bioflavonoid glucoside O-conjugates are absorbed from the intestine in the intact form or as their aglycones following hydrolysis by intestinal beta-glucosidases. In this study, the intestinal absorption of genistin, the beta-glucoside of the isoflavone genistein, was examined in anesthetized, adult female rats fitted with indwelling biliary cannulas. To first establish whether genistein, once absorbed, was converted into unique metabolites, genistin was infused into the femoral or portal veins and bile samples quantitatively collected. Analysis of bile samples by HPLC-mass spectrometry revealed that almost full recovery of the genistein component occurred in the form of unreacted genistin ( approximately 20%) and genistein 7beta-O-glucuronide ( approximately 80%). However, when genistin was infused into the upper small intestine, only genistein 7beta-O-glucuronide and the aglycone genistein appeared in the bile. There was no evidence for any biliary secretion of the unreacted genistin, thereby excluding its uptake in the intact form from the small intestine in this animal model.