Mutations of the novel renal glomerular genes NPHS1 and NPHS2 encoding nephrin and podocin cause two types of severe nephrotic syndrome presenting in early life, Finnish type congenital nephrotic syndrome (CNF) and a form of autosomal recessive familial focal segmental glomerulosclerosis (SRN1), respectively. To investigate the mechanisms by which mutations might cause glomerular protein leak, we analysed NPHS1/NPHS2 genotype/phenotype relationships in 41 non-Finnish CNF patients, four patients with congenital (onset 0 to 3 months) focal segmental glomerulosclerosis and five patients with possible SRN1 (onset 6 months to 2 years). We clarify the range of NPHS1 mutations in CNF, detecting mutation 'hot-spots' within the NPHS1 coding sequence. In addition, we describe a novel discordant CNF phenotype characterized by variable clinical severity, apparently influenced by gender. Moreover, we provide evidence that CNF may be genetically heterogeneous by detection of NPHS2 mutations in some CNF patients in whom NPHS1 mutations were not found. We confirm an overlap in the NPHS1/NPHS2 mutation spectrum with the characterization of a unique di-genic inheritance of NPHS1 and NPHS2 mutations, which results in a 'tri-allelic' hit and appears to modify the phenotype from CNF to one of congenital focal segmental glomerulosclerosis (FSGS). This may result from an epistatic gene interaction, and provides a rare example of multiple allelic hits being able to modify an autosomal recessive disease phenotype in humans. Our findings provide the first evidence for a functional inter-relationship between NPHS1 and NPHS2 in human nephrotic disease, thus underscoring their critical role in the regulation of glomerular filtration.

The leading causes of albuminuria and end-stage renal failure are secondary to abnormalities in the production or cellular action of insulin, including diabetes and hyperinsulinemic metabolic syndrome. The human glomerular podocyte is a critical cell for maintaining the filtration barrier of the kidney and preventing albuminuria. We have recently shown this cell to be insulin sensitive with respect to glucose uptake, with kinetics similar to muscle cells. We now show that the podocyte protein nephrin is essential for this process. Conditionally immortalized podocytes from two different patients with nephrin mutations (natural human nephrin mutant models) were unresponsive to insulin. Knocking nephrin down with siRNA in wild-type podocytes abrogated the insulin response, and stable nephrin transfection of nephrin-deficient podocytes rescued their insulin response. Mechanistically, we show that nephrin allows the GLUT1-and GLUT4-rich vesicles to fuse with the membrane of this cell. Furthermore, we show that the COOH of nephrin interacts with the vesicular SNARE protein VAMP2 in vitro and ex vivo (using yeast-2 hybrid and coimmunoprecipitation studies). This work demonstrates a previously unsuspected role of nephrin in vesicular docking and insulin responsiveness of podocytes.

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The transcriptional activity of nuclear receptors is mediated by coactivator proteins, including steroid receptor coactivator 1 (SRC1) and its homologues and the general coactivators CREB binding protein (CBP) and p300. SRC1 contains an activation domain (AD1) which functions via recruitment of CBP and and p300. In this study, we have used yeast two-hybrid and in vitro interaction-peptide inhibition experiments to map the AD1 domain of SRC1 to a 35-residue sequence potentially containing two ␣-helices. We also define a 72-amino-acid sequence in CBP necessary for SRC1 binding, designated the SRC1 interaction domain (SID). We show that in contrast to SRC1, direct binding of CBP to the estrogen receptor is weak, suggesting that SRC1 functions primarily as an adaptor to recruit CBP and p300. In support of this, we show that the ability of SRC1 to enhance ligand-dependent nuclear receptor activity in transiently transfected cells is dependent upon the integrity of the AD1 region. In contrast, the putative histone acetyltransferase domain, the Per-Arnt-Sim basic helix-loop-helix domain, the glutamine-rich domain, and AD2 can each be removed without loss of ligandinduced activity. Remarkably, a construct corresponding to residues 631 to 970, which contains only the LXXLL motifs and the AD1 region of SRC1, retained strong coactivator activity in our assays.The nuclear receptors (NRs) are ligand-regulated transcription factors that mediate the effects of steroids, retinoids, and other lipophilic hormones on gene expression (32). In common with other transcriptional activators, NRs stimulate transcription by promoting the local modification of chromatin structure and recruitment of a preinitiation complex (59). This is achieved via two transcriptional activation functions (AF1 and AF2) which provide molecular surfaces for the recruitment of transcriptional coactivator proteins (17,28,36,60).The AF2 surfaces of the ligand binding domains (LBDs) of NRs appear to be the principal sites for coactivator recruitment. Far-Western experiments detected two major classes of proteins in nuclear extracts (with apparent molecular masses of 160 and 140 kDa) which bind to the LBD of the estrogen receptor (ER) in the presence of ligand (5, 14). At least three distinct p160 proteins have been identified, including steroid receptor coactivator 1 (SRC1) (39), transcription intermediary factor 2 (TIF2) (54) and its murine homologue GRIP1 (18), and p300-CBP cointegrator-associated protein (pCIP) (50), which is the mouse homologue of the human protein AIB1 (1), also known as ACTR (8), RAC3 (29), or TRAM1 (49). These proteins appear to be bona fide coactivators, as they enhance the activity of NRs in both in vitro and in vivo experimental systems. The p140 class appears to consist chiefly of the nuclear protein RIP140 (6). The function of RIP140 is unknown, although it has been shown to down-regulate NR-mediated transcription in transient-reporter assays, possibly via competition with p160s for the LBD (15,27,35,51). Other AF2 binding proteins ...

An ␣-helical motif containing the sequence LXXLL is required for the ligand-dependent binding of transcriptional co-activators to nuclear receptors. By using a peptide inhibition assay, we have defined the minimal "core" LXXLL motif as an 8-amino acid sequence spanning positions ؊2 to ؉6 relative to the primary conserved leucine residue. In yeast two-hybrid assays, core LXXLL motif sequences derived from steroid receptor co-activator (SRC1), the 140-kDa receptor interacting protein (RIP140), and CREB-binding protein (CBP) displayed differences in selectivity and affinity for nuclear receptor ligand binding domains. Although core LXXLL motifs from SRC1 and RIP140 mediated strong interactions with steroid and retinoid receptors, three LXXLL motifs present in the global co-activator CBP were found to have very weak affinity for these proteins. Core motifs with high affinity for steroid and retinoid receptors were generally found to contain a hydrophobic residue at position ؊1 relative to the first conserved leucine and a nonhydrophobic residue at position ؉2. Our results indicate that variant residues in LXXLL core motifs influence the affinity and selectivity of co-activators for nuclear receptors.Ligand-dependent gene expression mediated by nuclear receptors involves the recruitment of transcriptional co-activators to the ligand binding domain (LBD).1 The LBD is structurally conserved among the NR family and consists of between 10 and 12 ␣-helices folded in a globular domain (1). A central hydrophobic pocket accommodates the cognate ligand, which upon binding induces a conformational change in the LBD, exposing a co-activator docking site on the LBD surface. Sequence conservation, mutational analyses, and crystal structures have indicated that the co-activator docking site is made up of residues from helices 3, 5, and 12, which form a hydrophobic channel conserved among the NR family members. The integrity of this surface is essential for co-activator binding and as a consequence the transactivation function (AF-2) of the LBD (reviewed in Ref.2).We have previously shown that a short ␣-helical sequence, the LXXLL motif, is necessary and sufficient for mediating the interaction of the co-activators RIP140, SRC1 and CBP/p300 with NRs (3). LXXLL motifs have also been shown to mediate binding of other co-activators including TIF1 (4), TIF2/GRIP1 (5, 6), p300/CBP-interacting protein/ACTR (7, 8), TRAP220/ DRIP205 (9 -11), PPAR␥ co-activator-1 (12), and ASC-2/ RAP250/NRC (13-15) to NRs. The number and sequence of LXXLL motifs varies considerably among the co-activators and is likely to account for observed differences in binding of coactivators to selected NRs or classes of NRs. The 160-kDa co-activators represented by SRC1, TIF2, and p/CIP each have a nuclear receptor interaction domain (NID) containing three LXXLL motifs. These sequences and the spacing between them are highly conserved, and we and others (3,5,7,16,17) have shown that they mediate high affinity binding to NRs. In addition, the SRC1a isoform has an addi...