New insights into potential functions for the protein 4.1superfamily of proteins in kidney epithelium Page: 4 of 32
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Protein 4.1 in kidney physio-pathology
kidney epithelium, respectively (Figure 4). This specialized epithelial organization may play an important role in coordination of
ion transport. Indeed the level of activity of NHE3 at the apical pole of the TAL epithelium has been demonstrated to depend on
the level of activity of NHE1 at the basolateral pole (175-177), the integrity of the cytoskeleton being required for this functional
link to operate (178). The potential relevance of this intriguing distribution in control of cell proliferation will be discussed later
in this chapter.
4.3. Renal ankyrins
Structure and function of renal ankyrins are discussed in another chapter of this issue. We will only emphasize here
that, like renal 4.1 proteins: i) renal ankyrins are expressed as products of distinct ankyrin genes at the basolateral pole of the
kidney epithelium; ii) these ankyrin gene products actually correspond to various isoforms generated by tissue-specific pre-
mRNA splicing events; iii) renal ankyrins show mutually exclusive expression along the nephron; and iv) renal ankyrins mediate
proper anchorage of key ion transporters and cell adhesion molecules in the basolateral plasma membrane of the kidney
5. POTENTIAL FUNCTIONS FOR RENAL 4.1 PROTEINS
As a first step to decipher the roles of 4.1 proteins in kidney structure and function, we have begun to identify potential
binding partners for kidney 4.1B, kidney 4.iR and kidney 4.iN, through the screening of a rat kidney yeast two-hybrid system
cDNA library with full length renal 4.1 proteins (71) and 4.iR CTD baits.
A rat kidney yeast two-hybrid system cDNA library, cloned into the GAL4 activation domain vector pGAD3S-2X
(179, 180), was screened for binding partners using baits corresponding to cDNAs encoding full length coding regions of the
major kidney-specific isoforms of mouse 4.iN, 4.iR and 4.1B (71), or the C-terminal domain of mouse 4.iR. Baits were cloned
into the LexA DNA binding domain vector pLEX12 (179, 180) and used for yeast transformation. Yeast transformed with each
pLEX12 bait cDNA, was selected in absence of Tryptophane, then transformed with the pGAD3S-2X rat kidney cDNA library
conferring upon yeast growth in absence of Leucine. Yeast was grown on triple selection plates lacking Tryptophane, Leucine
and Histidine in order to select clones in which the bait of interest and putative preys interacted with each other. Such protein-
protein interactions restore a fully active GAL4 transcription factor and drive the expression of histidine selection and f3-
galactosidase reporter genes. Selected clones were further screened for prey interaction with kidney 4.1 protein baits based on
standard X-galactose filter assay (179, 180). f -galactosidase positive clones were then characterized by DNA sequencing using a
forward pGAD3 S-2X vector specific primer. Interactions were re-confirmed in yeast co-transformed with cDNAs coding for the
bait and the prey of interest. Further mapping of the regions in 4.1 proteins responsible for the identified interactions was
determined after probing preys with various truncated variants of protein 4.1 FERM and CTD domains. Only relevant clones (i.e.
with prey coding sequences in frame) are presented below.
5.2. Identification of binding partners for renal 4.1 proteins
The results of our screen to date are summarized in Table I. The predominant binding partners for kidney 4.1B (27/32
clones) and exclusive binding partners for kidney 4.1N (17/17 clones) correspond to three members of the 14-3-3 family of
proteins: 14-3-3theta , 14-3-3zeta , and 14-3-3beta. In contrast, these proteins represent a minor category of binding partners for
kidney 4.iR (3/16 clones). Another binding partner for kidney 4.1B (5/32 clones) is pICln, a cell swelling-activated chloride
channel previously reported to interact with 4.iR (46). Additional potential binding partners for kidney 4.iR include: a putative
phosphatase suspected to promote cell proliferation, LRP16 (6/16 clones) (181, 182); two proteins involved in trafficking,
SEC14L1 (1/16 clones), a mammalian homolog of yeast phosphatidylinositol/phosphatidylcholine transfer protein SEC14 (183)
and the Rab GDP-dissociation inhibitor (GDI) Rab-GDIalpha (2/16 clones), a regulatory protein for small GTP-binding Rab
proteins that regulates vesicle-mediated cellular transport through control of Rab GDP/GTP exchange reaction and translocation
of Rab proteins between the cytosol and cell membranes (184-190). Of particular note, a small GTPase of the Rab family has
been recently shown to regulate postsynaptic terminal trafficking of AMPA receptors (191), a class of receptors that has been
recently reported to interact with 4.iN and PDZ domain-containing protein SAP 97 (20). Another 4.1R binding partner identified
is the beta-Amyloid Precursor Protein (beta-APP) (1/16 clones), a key protein in Alzheimer's disease (AD) pathogenesis (192).
4.iR may potentially bind to a component of the protein translation machinery involved in cytoskeleton reorganization and cell
transformation, i.e. elongation initiation factor lalpha (2/16 clones) (193, 194). Lastly, we identified the putative tumor
suppressor TMEM24 (195) (1/16 clones), a gene present in the l1q23.3 locus and frequently deleted in neuroblastomas, as a
potential binding partner for 4.iR.
While it will be important to confirm the various protein-protein interactions identified by other methodological
approaches, some of them have been previously documented in the literature. As such, we will focus our discussion on the
following three classes of binding partners: beta-APP, 14-3-3 proteins, and the cell swelling-activated chloride channel pICln.
5.3. J-Amyloid Precursor Protein: a key element in progression of neuropathies and potentially epitheliopathies
Progressive cerebral deposition of the amyloid beta-peptide (A-beta peptide) is an early and invariant feature of
Alzheimer's disease (196). This peptide originates from proteolytic cleavage of the very C-terminal region of the beta-amyloid
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Calinisan, Venice; Gravem, Dana; Chen, Ray Ping-Hsu; Brittin,Sachi; Mohandas, Narla; Lecomte, Marie-Christine et al. New insights into potential functions for the protein 4.1superfamily of proteins in kidney epithelium, article, June 17, 2005; Berkeley, California. (digital.library.unt.edu/ark:/67531/metadc892527/m1/4/: accessed October 20, 2018), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT Libraries Government Documents Department.