Regulation of albumin endocytosis by PSD95/Dlg/ZO-1 (PDZ) scaffolds - Interaction of Na+-H+ exchange regulatory factor-2 with ClC-5

The constitutive reuptake of albumin from the glomerular filtrate by receptor-mediated endocytosis is a key function of the renal proximal tubules. Both the Cl- channel ClC-5 and the Na+-H+ exchanger isoform 3 are critical components of the macromolecular endocytic complex that is required for albumin uptake, and therefore the cell-surface levels of these proteins may limit albumin endocytosis. This study was undertaken to investigate the potential roles of the epithelial PDZ scaffolds, Na+-H+ exchange regulatory factors, NHERF1 and NHERF2, in albumin uptake by opossum kidney ( OK) cells. We found that ClC-5 co-immunoprecipitates with NHERF2 but not NHERF1 from OK cell lysate. Experiments using fusion proteins demonstrated that this was a direct interaction between an internal binding site in the C terminus of ClC-5 and the PDZ2 module of NHERF2. In OK cells, NHERF2 is restricted to the intravillar region while NHERF1 is located in the microvilli. Silencing NHERF2 reduced both cell-surface levels of ClC-5 and albumin uptake. Conversely, silencing NHERF1 increased cell-surface levels of ClC-5 and albumin uptake, presumably by increasing the mobility of NHE3 in the membrane and its availability to the albumin uptake complex. Surface biotinylation experiments revealed that both NHERF1 and NHERF2 were associated with the plasma membrane and that NHERF2 was recruited to the membrane in the presence of albumin. The importance of the interaction between NHERF2 and the cytoskeleton was demonstrated by a significant reduction in albumin uptake in cells overexpressing an ezrin binding-deficient mutant of NHERF2. Thus NHERF1 and NHERF2 differentially regulate albumin uptake by mechanisms that ultimately alter the cell-surface levels of ClC-5.

Postnatal developmental expression of the PDZ scaffolds Na+-H+ exchanger regulatory factors 1 and 2 in the rat cochlea

Sensory transduction in the mammalian cochlea requires the maintenance of specialized fluid compartments with distinct ionic compositions. This is achieved by the concerted action of diverse ion channels and transporters, some of which can interact with the PDZ scaffolds, Na+-H+ exchanger regulatory factors 1 and 2 (NHERF-1, NHERF-2). Here, we report that NHERF-1 and NHERF-2 are widely expressed in the rat cochlea, and that their expression is developmentally regulated. Reverse transcription/polymerase chain reaction (RT-PCR) and Western blotting initially confirmed the RNA and protein expression of NHERFs. We then performed immunohistochemistry on cochlea during various stages of postnatal development. Prior to the onset of hearing (P8), NHERF-1 immunolabeling was prominently polarized to the apical membrane of cells lining the endolymphatic compartment, including the stereocilia and cuticular plates of the inner and outer hair cells, marginal cells of the stria vascularis, Reissner's epithelia, and tectorial membrane. With maturation (P21, P70), NHERF-1 immunolabeling was reduced in the above structures, whereas labeling increased in the apical membrane of the interdental cells of the spiral limbus and the inner and outer sulcus cells, Hensen's cells, the inner and outer pillar cells, Deiters cells, the inner border cells, spiral ligament fibrocytes, and spiral ganglion neurons (particularly type II). NHERF-1 expression in strial basal and intermediate cells was persistent. NHERF-2 immunolabeling was similar to that for NHERF-1 during postnatal development, with the exception of expression in the synaptic regions beneath the outer hair cells. NHERF-1 and NHERF-2 co-localized with glial fibrillary acidic protein and vimentin in glia. The cochlear localization of NHERF scaffolds suggests that they play important roles in the developmental regulation of ion transport, homeostasis, and auditory neurotransmission.

Cloning and expression of the large zebrafish protocadherin gene, Fat

The cadherin superfamily members play an important role in mediating cell-cell contact and adhesion (Takeichi, M., 1991. Cadherin cell adhesion receptors as a morphogenetic regulator. Science 251, 1451-1455). A distinct subfamily, neither belonging to the classical or protocadherins includes Fat, the largest member of the cadherin super-family. Fat was originally identified in Drosophila. Subsequently, orthologues of Fat have been described in man (Dunne, J., Hanby, A. M., Poulsom, R., Jones, T. A., Sheer, D., Chin, W. G., Da, S. M., Zhao, Q., Beverley, P. C., Owen, M. J., 1995. Molecular cloning and tissue expression of FAT, the human homologue of the Drosophila fat gene that is located on chromosome 4q34-q35 and encodes a putative adhesion molecule. Genomics 30, 207-223), rat (Ponassi, M., Jacques, T. S., Ciani, L., ffrench, C. C., 1999. Expression of the rat homologue of the Drosophila fat tumour suppressor gene. Mech. Dev. 80, 207-212) and mouse (Cox, B., Hadjantonakis, A. K., Collins, J. E., Magee, A. I., 2000. Cloning and expression throughout mouse development of mfat 1, a homologue of the Drosophila tumour suppressor gene fat [In Process Citation]. Dev. Dyn. 217, 233-240). In Drosophila, Fat has been shown to play an important role in both planar cell polarity and cell boundary formation during development. In this study we describe the characterization of zebrafish Fat, the first non-mammalian, vertebrate Fat homologue to be identified. The Fat protein has 64% amino acid identity and 80% similarity to human FAT and an identical domain structure to other vertebrate Fat proteins. During embryogenesis fat mRNA is expressed in the developing brain, specialised epithelial surfaces the notochord, ears, eyes and digestive tract, a pattern similar but distinct to that found in mammals. (c) 2005 Elsevier B.V. All rights reserved.