CLIC5A, a component of the ezrin-podocalyxin complex in glomeruli, is a determinant of podocyte integrity.

The chloride intracellular channel 5A (CLIC5A) protein, one of two isoforms produced by the CLIC5 gene, was isolated originally as part of a cytoskeletal protein complex containing ezrin from placental microvilli. Whether CLIC5A functions as a bona fide ion channel is controversial. We reported previously that a CLIC5 transcript is enriched approximately 800-fold in human renal glomeruli relative to most other tissues. Therefore, this study sought to explore CLIC5 expression and function in glomeruli. RT-PCR and Western blots show that CLIC5A is the predominant CLIC5 isoform expressed in glomeruli. Confocal immunofluorescence and immunogold electron microscopy reveal high levels of CLIC5A protein in glomerular endothelial cells and podocytes. In podocytes, CLIC5A localizes to the apical plasma membrane of foot processes, similar to the known distribution of podocalyxin and ezrin. Ezrin and podocalyxin colocalize with CLIC5A in glomeruli, and podocalyxin coimmunoprecipitates with CLIC5A from glomerular lysates. In glomeruli of jitterbug (jbg/jbg) mice, which lack the CLIC5A protein, ezrin and phospho-ERM levels in podocytes are markedly lower than in wild-type mice. Transmission electron microscopy reveals patchy broadening and effacement of podocyte foot processes as well as vacuolization of glomerular endothelial cells. These ultrastructural changes are associated with microalbuminuria at baseline and increased susceptibility to adriamycin-induced glomerular injury compared with wild-type mice. Together, the data suggest that CLIC5A is required for the development and/or maintenance of the proper glomerular endothelial cell and podocyte architecture. We postulate that the interaction between podocalyxin and subjacent filamentous actin, which requires ezrin, is compromised in podocytes of CLIC5A-deficient mice, leading to dysfunction under unfavorable genetic or environmental conditions.

Characterization and prevalence of PITX2 microdeletions and mutations in Axenfeld-Rieger malformations.

PURPOSE: Mutations of the homeodomain protein PITX2 produce Axenfeld-Rieger (AR) malformations of the anterior chamber, an autosomal dominant disorder accompanied by a 50% risk of glaucoma. Twenty-nine mutations of PITX2 have been described, with a mutational prevalence estimated between 10% and 60% in AR. In the current study, the possible role of altered PITX2 gene dosage in the etiology of AR was investigated. Gross gene deletions and duplications should alter PITX2 activity analogously to hypomorphic and hypermorphic mutations, respectively. METHODS: Sixty-four patients with AR, iridogoniodysgenesis (IGD), iris hypoplasia (IH), or anterior segment dysgenesis (ASD) were screened for PITX2 mutations by sequencing. PITX2 gene dosage was concurrently examined in these patients by real-time quantitative PCR. Microsatellite markers were used to map 4q25 microdeletions at a contig scale, as well as for haplotype analysis in an extended AR kindred. An additional 27 patients with other assorted ocular phenotypes were evaluated by similar methods, amounting to a total of 91 cases analyzed. RESULTS: Three novel mutations of PITX2 (4.7%) were identified among 64 patients with AR, IGD, IH, or ASD. Deletions of PITX2 were as frequent as mutations in our sample. Chromosome 4q25 microdeletions were physically mapped relative to several microsatellite markers in each patient. Cosegregation of AR and a PITX2 deletion was demonstrated in an extended kindred. CONCLUSIONS: Point mutations and gross deletions of PITX2 appear to produce an equivalent haploinsufficiency phenotype. Quantitative PCR is an efficient means of detecting causative PITX2 deletions in patients with AR and may increase the detection rate at this locus.

Analysis of mutations of the PITX2 transcription factor found in patients with Axenfeld-Rieger syndrome.

PURPOSE: To assess the effects of previously uncharacterized PITX2 missense mutations found in patients with Axenfeld-Rieger syndrome and to determine the functional roles of the C-terminal region of PITX2. METHODS: Recombinant PITX2 proteins were analyzed with the use of cellular immunofluorescence, electrophoretic mobility shift, reporter transactivation, and protein half-life assays in human trabecular meshwork cells. RESULTS: Two homeobox mutations, R43W and R90C, resulted in severely reduced DNA-binding and transcriptional activation despite normal nuclear localization. L105V, located C-terminal to the homeodomain, resulted in normal localization, reporter gene transactivation, and protein half-life, but with an altered mobility shift pattern of protein-DNA complexes. N108T, also located C-terminal to the homeodomain, resulted in an altered mobility shift pattern and with slightly increased reporter transactivation and shortened protein half-life. The PITX2 C-terminal region contains at least three domains, each with distinct modulating effects on reporter transactivation. CONCLUSIONS: PITX2 homeobox mutations predictably resulted in decreased function of the protein. However, the two C-terminal mutations exhibited only subtle defects on PITX2 transactivation and protein-DNA binding, suggesting that ocular development is sensitive to even slight alterations of PITX2 function. The C-terminal mutations L105V and N108T lie in a domain that inhibits PITX2 transcriptional activation. These two mutations produce electrophoretic mobility shift assay patterns representing altered protein-DNA interactions that may be important for accurate target gene selection. Additionally, N108T resulted in a less stable PITX2 mutant protein with elevated activity that may result in stochastic dysregulation during critical stages of development. Together, the results clearly indicate that stringent control of PITX2 is required for normal ocular development and function.

Phosphorylation of TIMAP by glycogen synthase kinase-3beta activates its associated protein phosphatase 1.

TIMAP (TGF-beta1 inhibited, membrane-associated protein) is a prenylated, endothelial cell-predominant protein phosphatase 1 (PP1c) regulatory subunit that localizes to the plasma membrane of filopodia. Here, we determined whether phosphorylation regulates TIMAP-associated PP1c function. Phosphorylation of TIMAP was observed in cells metabolically labeled with [32P]orthophosphate and was reduced by inhibitors of protein kinase A (PKA) and glycogen synthase kinase-3 (GSK-3). In cell-free assays, immunopurified TIMAP was phosphorylated by PKA and, after PKA priming, by GSK-3beta. Site-specific Ser to Ala substitution identified amino acid residues Ser333/Ser337 as the likely PKA/GSK-3beta phosphorylation site. Substitution of Ala for Val and Phe in the KVSF motif of TIMAP (TIMAPV64A/F66A) abolished PP1c binding and TIMAP-associated PP1c activity. TIMAPV64A/F66A was hyper-phosphorylated in cells, indicating that TIMAP-associated PP1c auto-dephosphorylates TIMAP. Constitutively active GSK-3beta stimulated phosphorylation of TIMAPV64A/F66A, but not wild-type TIMAP, suggesting that the PKA/GSK-3beta site may be subject to dephosphorylation by TIMAP-associated PP1c. Substitution of Asp or Glu for Ser at amino acid residues 333 and 337 to mimic phosphorylation reduced the PP1c association with TIMAP. Conversely, GSK-3 inhibitors augmented PP1c association with TIMAP-PP1c in cells. The 333/337 phosphomimic mutations also increased TIMAP-associated PP1c activity in vitro and against the non-integrin laminin receptor 1 in cells. Finally, TIMAP mutants with reduced PP1c activity strongly stimulated endothelial cell filopodia formation, an effect mimicked by the GSK-3 inhibitor LiCl. We conclude that TIMAP is a target for PKA-primed GSK-3beta-mediated phosphorylation. This phosphorylation controls TIMAP association and activity of PP1c, in turn regulating extension of filopodia in endothelial cells.

The protein phosphatase-1 targeting subunit TIMAP regulates LAMR1 phosphorylation.

TIMAP is a prenylated endothelial cell protein with a domain structure that predicts it to be a protein phosphatase-1 (PP-1) regulatory subunit. We found that TIMAP interacts with the 37/67 kDa laminin receptor (LAMR1) in yeast two-hybrid assays. In endothelial cells, endogenous TIMAP and LAMR1 co-immunoprecipitated and co-localized at the plasma membrane. TIMAP amino acids 261-290, representing the fourth ankyrin repeat of TIMAP, are necessary and sufficient for the interaction. In MDCK cells, lacking endogenous TIMAP, overexpression of full-length TIMAP, but not TIMAP deleted in the fourth ankyrin domain, allowed co-immunoprecipitation with LAMR1. PP-1 co-precipitated with overexpressed and endogenous TIMAP in MDCK and endothelial cells, respectively. In MDCK cells, PP-1 associated with LAMR1 in the presence, but not in the absence, of TIMAP. LAMR1 was a substrate for PP-1 in vitro, and in MDCK cells its phosphorylation was abrogated by expression of full-length TIMAP but not by TIMAP deficient in the fourth ankyrin domain. Hence, TIMAP targets PP-1 to LAMR1, and LAMR1 is a TIMAP-dependent PP-1 substrate.

Molecular genetics of Axenfeld-Rieger malformations.

Axenfeld-Rieger (AR) malformations are autosomal dominant developmental defects of the anterior segment of the eye, and often result in glaucomatous blindness. AR malformations are associated with mutations in two transcription factor genes (PITX2 and FOXC1) expressed throughout eye ontogeny. Studies of disease-associated mutant proteins have provided insights into the aetiology of AR malformations, while delineating residues and domains important to DNA binding, transactivation and nuclear localization. The availability of mouse models for both PITX2 and FOXC1 has allowed detailed study of their expression and mutant phenotypes. Dissection of the normal functions and domain structures of these factors will aid in future elucidation of how alterations of the developmental program produce the dysgenic phenotypes seen in AR. There are at least two AR loci still awaiting molecular cloning on chromosomes 13q14 and 16q24. Identification of further genes implicated in aberrations of human ocular development will advance our understanding of the mechanisms whereby pattern is established in the eye, and may be of clinical value in treating the glaucoma that is the most serious consequence of AR malformations.