Ann L. Hubbard

Copper handling machinery of the brain

Copper plays an indispensable role in the physiology of the human central nervous system (CNS). As a cofactor of dopamine-β-hydroxylase, peptidyl-α-monooxygenase, superoxide dismutases, and many other enzymes, copper is a critical contributor to catecholamine biosynthesis, activation of neuropeptides and hormones, protection against reactive oxygen species, respiration and other processes essential for normal CNS function. Copper content in the CNS is tightly regulated, and changes in copper levels in the brain are associated with a wide spectrum of pathologies. However, the mechanistic understanding of copper transport in the CNS is still in its infancy. Little is known about copper distribution among various cell types or cell-specific regulation of copper homeostasis, despite the fact that the molecules mediating copper transport and distribution in the brain (CTR1, Atox1, CCS, ScoI/II, ATP7A and ATP7B) have been identified and their importance in CNS function increasingly understood. In this review, we summarize current knowledge about copper levels and uses in the CNS and describe the molecules involved in maintaining copper homeostasis in the brain.

Scribble associates with two polarity proteins, Lgl2 and Vangl2, via distinct molecular domains

Scribble (Scrib) is a large multi‐domain cytoplasmic protein that was first identified through its requirement for the establishment of epithelial polarity. We tested the hypotheses that Scrib asssociates with the basolateral membrane via multiple domains, binds specific protein partners, and is part of a multimeric complex. We generated a series of EGFP‐tagged Scrib fusion proteins and examined their membrane localizations in two types of polarized mammalian epithelial cells using biochemical and morphological approaches. We found that Scribs Leucine‐rich‐repeat (LRR) and PDS‐95/Discs Large/ZO‐1 (PDZ) domains independently associate with the plasma membrane in both cell types. We identified multiple large Scrib complexes, demonstrated that Scrib and the cytoplasmic protein Lethal giant larvae2 (Lgl2) co‐IP and that this association occurs via Scribs LRR domain. Further, this report demonstrates that the membrane protein Vangl2 binds selectively to specific PDZ domains in Scrib. Our identification of Scribs associations highlights its function in multiple biologic pathways and sets the stage for future identification of more proteins that must interact with Scribs remaining domains. J. Cell. Biochem. 99: 647–664, 2006.

Plasma membrane protein sorting in epithelial cells: Do secretory pathways hold the key?

Abstract The Madin-Darby canine kidney (MDCK) cell sorts newly synthesized apical and basolateral plasma membrane proteins intracellularly and then ships them directly to the correct plasma membrane domains, whereas the rat hepatocyte sorts apical and basolateral proteins after their arrival at the basolateral domain of the plasma membrane and then employs a transcytotic mechanism to deliver the apical proteins to the apical domain. This difference in plasma membrane protein sorting pathways may relate to the observation that the hepatocyte, unlike the MDCK cell, does not have an apically directed secretory pathway.

Hepatocyte plasma membrane ecto-ATPase ( pp120 HA4) is a substrate for tyrosine kinase activity of the insulin receptor

pp120/HA4, a membrane protein found in hepatocyte plasma membranes and a substrate for the insulin receptor tyrosine kinase, was purified to homogeneity, subjected to partial proteolysis, and peptides were sequenced by Edman degradation. Six amino acid sequences were obtained, and they matched the deduced amino acid sequences of six regions of a hepatocyte membrane protein called ecto-ATPase.

A Novel Cellular Phenotype for Familial Hypercholesterolemia due to a Defect in Polarized Targeting of LDL Receptor

Basolateral targeting of membrane proteins in polarized epithelial cells typically requires cytoplasmic domain sorting signals. In the familial hypercholesterolemia (FH)-Turku LDL receptor allele, a mutation of glycine 823 residue affects the signal required for basolateral targeting in MDCK cells. We show that the mutant receptor is mistargeted to the apical surface in both MDCK and hepatic epithelial cells, resulting in reduced endocytosis of LDL from the basolateral/sinusoidal surface. Consequently, virally encoded mutant receptor fails to mediate cholesterol clearance in LDL receptor-deficient mice, suggesting that a defect in polarized LDL receptor expression in hepatocytes underlies the hypercholesterolemia in patients harboring this allele. This evidence directly links the pathogenesis of a human disease to defects in basolateral targeting signals, providing a genetic confirmation of these signals in maintaining epithelial cell polarity.

Newly synthesized hepatocyte plasma membrane proteins are transported in transcytotic vesicles in the bile duct-ligated rat

BACKGROUND Newly synthesized apical membrane proteins in hepatocytes go first to the basolateral membrane, from which they are retrieved and delivered to the apical domain. The goal of the present study was to identify the vesicular carriers of these molecules. METHODS The common bile duct of rats was ligated for 10-72 hours, and then various plasma membrane proteins were localized using immunofluorescence and quantitative immuno-electron microscopy of fixed liver tissue. RESULTS By immunofluorescence, we found intracellular punctate staining near the bile canalicular membrane of polymeric immunoglobulin A (IgA) receptor and several apical membrane proteins, but not basolateral proteins. This compartment was membrane bounded and pleiomorphic by immunoelectron microscopy. Colocalization at the electron microscopic level showed that the apical protein, dipeptidyl peptidase IV, was in the same structures as aminopeptidase N, polymeric IgA receptor, or intravenously injected horseradish peroxidase. This intracellular immunolabeling decreased after cycloheximide treatment (t1/2 = 2-2.5 hours) or reversal of the ligation for 1 hour. In the latter case, bile canalicular labeling increased. Furthermore, polymeric IgA receptor was delivered to the bile canaliculi. CONCLUSIONS Bile duct ligation leads to an intracellular accumulation of vesicles carrying polymeric IgA receptor, several apical membrane proteins, and a fluid phase marker. These vesicles continue to fuse with the apical membrane, even during ligation.

Tissue-specific Regulation of Sodium/Proton Exchanger Isoform 3 Activity in Na+/H+ Exchanger Regulatory Factor 1 (NHERF1) Null Mice cAMP INHIBITION IS DIFFERENTIALLY DEPENDENT ON NHERF1 AND EXCHANGE PROTEIN DIRECTLY ACTIVATED BY cAMP IN ILEUM VERSUS PROXIMAL TUBULE

The multi-PDZ domain containing protein Na+/H+ Exchanger Regulatory Factor 1 (NHERF1) binds to Na+/H+ exchanger 3 (NHE3) and is associated with the brush border (BB) membrane of murine kidney and small intestine. Although studies in BB isolated from kidney cortex of wild type and NHERF1-/- mice have shown that NHERF1 is necessary for cAMP inhibition of NHE3 activity, a role of NHERF1 in NHE3 regulation in small intestine and in intact kidney has not been established. Here a method using multi-photon microscopy with the pH-sensitive dye SNARF-4F (carboxyseminaphthorhodafluors-4F) to measure BB NHE3 activity in intact murine tissue and use it to examine the role of NHERF1 in regulation of NHE3 activity. NHE3 activity in wild type and NHERF1-/- ileum and wild type kidney cortex were inhibited by cAMP, whereas the cAMP effect was abolished in kidney cortex of NHERF1-/- mice. cAMP inhibition of NHE3 activity in these two tissues is mediated by different mechanisms. In ileum, a protein kinase A (PKA)-dependent mechanism accounts for all cAMP inhibition of NHE3 activity since the PKA antagonist H-89 abolished the inhibitory effect of cAMP. In kidney, both PKA-dependent and non-PKA-dependent mechanisms were involved, with the latter reproduced by the effect on an EPAC (exchange protein directly activated by cAMP) agonist (8-(4-chlorophenylthio)-2′O-Me-cAMP). In contrast, the EPAC agonist had no effect in proximal tubules in NHERF1-/- mice. These data suggest that in proximal tubule, NHERF1 is required for all cAMP inhibition of NHE3, which occurs through both EPAC-dependent and PKA-dependent mechanisms; in contrast, cAMP inhibits ileal NHE3 only by a PKA-dependent pathway, which is independent of NHERF1 and EPAC.

The establishment of hepatocyte cell surface polarity during fetal liver development

Antibodies to six glycoproteins present in different domains of the hepatocyte plasma membrane were used to study the establishment of cell surface polarity during rat fetal liver development. The proteins were immunoprecipitated from fetal liver homogenates between 14 and 21 days of gestation and quantified by immunoblotting. Aminopeptidase N, CE 9, and HA 321, which reside in the apical, basolateral, and lateral plasma membrane in the adult hepatocyte, respectively, were present in high concentrations at 14 days of gestation and remained high until birth. In contrast, two apical proteins (HA 4 and dipeptidyl peptidase IV) and two basolateral proteins (ASGP receptor and EGF receptor) were first detected between 16 and 18 days of gestation and increased linearly until birth. HA 4 was the only molecule for which the fetal and adult forms differed, with the former having a faster mobility on SDS-PAGE, due to differences in N-linked oligosaccharides. With two exceptions, the localization of the molecules from earliest detection was restricted to the same domain as that in the adult. At 15 days of gestation, HA 321 and a small portion of aminopeptidase were detected on the basolateral membrane. By 21 days both molecules had assumed their adult localization pattern. Our results indicate that the biogenesis of cell surface polarity is an early event, implying that the mechanisms for sorting plasma membrane molecules are functional very early in development. Furthermore, the different patterns of appearance of the six molecules, irrespective of domain, indicate that the biochemical composition of the cell surface changes dramatically during fetal liver development.

Defective jejunal and colonic salt absorption and alteredNa +/H+ exchanger 3 (NHE3) activity in NHE regulatory factor 1 (NHERF1) adaptor protein-deficient mice

We investigated the role of the Na+/H+ exchanger regulatory factor 1 (NHERF1) on intestinal salt and water absorption, brush border membrane (BBM) morphology, and on the NHE3 mRNA expression, protein abundance, and transport activity in the murine intestine. NHERF1-deficient mice displayed reduced jejunal fluid absorption in vivo, as well as an attenuated in vitro Na+ absorption in isolated jejunal and colonic, but not of ileal, mucosa. However, cAMP-mediated inhibition of both parameters remained intact. Acid-activated NHE3 transport rate was reduced in surface colonocytes, while its inhibition by cAMP and cGMP was normal. Immunodetection of NHE3 revealed normal NHE3 localization in the BBM of NHERF1 null mice, but NHE3 abundance, as measured by Western blot, was significantly reduced in isolated BBM from the small and large intestines. Furthermore, the microvilli in the proximal colon, but not in the small intestine, were significantly shorter in NHERF1 null mice. Additional knockout of PDZK1 (NHERF3), another member of the NHERF family of adaptor proteins, which binds to both NHE3 and NHERF1, further reduced basal NHE3 activity and caused complete loss of cAMP-mediated NHE3 inhibition. An activator of the exchange protein activated by cAMP (EPAC) had no effect on jejunal fluid absorption in vivo, but slightly inhibited NHE3 activity in surface colonocytes in vitro. In conclusion, NHERF1 has segment-specific effects on intestinal salt absorption, NHE3 transport rates, and NHE3 membrane abundance without affecting mRNA levels. However, unlike PDZK1, NHERF1 is not required for NHE3 regulation by cyclic nucleotides.

125I-wheat germ agglutinin blotting: increased sensitivity with polyvinylpyrrolidone quenching and periodate oxidation/reductive phenylamination.

Two substantial improvements in sensitivity in the identification of 125I-wheat germ agglutinin-binding glycoproteins on nitrocellulose blots of sodium dodecyl sulfate-polyacrylamide gels are reported. The major improvement in sensitivity (about 30-fold) derives from the use of 2% (w/v) polyvinylpyrrolidone (average Mr 40,000) instead of bovine serum albumin or denatured hemoglobin as the quenching agent (or carrier) during incubation with 125I-wheat germ agglutinin in detergent-free, phosphate-buffered saline. Under these conditions, specific labeling with 125I-wheat germ agglutinin is observed for orosomucoid derivatives that display N-acetylglucosamine or sialic acid residues at the nonreducing termini of their oligosaccharides, as well as for a number of glycoprotein components of a rat hepatocyte plasma membrane fraction. An additional improvement in sensitivity (up to 10-fold) results from an increase in the binding of 125I-wheat germ agglutinin to sialic acid-containing glycoproteins after treatment of the blots with 5 mM sodium metaperiodate followed by 5 mM aniline in the presence of 30 mM sodium cyanoborohydride. This treatment appears to cause the sequential oxidation and reductive phenylamination of the side chain of glycoprotein sialic acid residues.