Abstract: TH-PO385

FRMD3/Protein 4.1O Is a Novel Nephrin Adaptor to F-Actin Regulated by MAPK- and Src-Kinases and Linked to Diabetes

Session Information

Category: Cell Biology

  • 201 Cell Signaling, Oxidative Stress

Authors

  • Koenigshausen, Eva, Medical Faculty Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany
  • Sellin, Lorenz, Medical Faculty Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany
  • Bajraktarevic, Aida, Medical Faculty Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany
  • Ohlsson, Sinja, Medical Faculty Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany
  • Stahl, Klaus, Hannover Medical School, Hannover, Germany
  • Schönberger, Marie, Medical Faculty Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany
  • Wiech, Thorsten, Department of Pathology, University Hospital Hamburg Eppendorf, Hamburg, Germany
  • Haller, Hermann G., Hannover Medical School, Hannover, Germany
  • Rump, Lars C., Medical Faculty Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany
  • Schiffer, Mario, Hannover Medical School, Hannover, Germany
Background

FRMD3 has been proposed as a candidate gene for diabetic nephropathy (DN). FRMD3 encodes for protein 4.1O, a member of the 4.1 protein family. In erythrocytes, protein 4.1R links membrane proteins to the actin cytoskeleton. The molecular function of protein 4.1O is unknown so far.

Methods

Expression and interaction of protein 4.1O were investigated by qPCR,IF and western blot in podocytes. Zebrafish larvae were treated with morpholinos against moe the orthologue of FRMD3. The loss of 78 kD-GFP tagged protein using the Tg(I-fab:DBP-eGFP) fish line was measured. Human 4.1O truncations were reexpressed in moe knockdown zebrafish larvae. Electronmicroscopy was performed. Cells expressing protein 4.1O, its truncations, its point mutations and nephrin or nck were subjected to co-immunoprecipitation. Cells were incubated with kinase inhibitors PP2 (10 μM) and SB202190 (50 μM). Kidney samples from patients with T1DN or T2DN and from streptozotocin treated mice were stained for protein 4.1O.

Results

Protein 4.1O is expressed in human podocytes and interacts with nephrin, GLEPP1, IQGAP1, Neph1 and actin in vitro and in vivo. Injection of moe morpholinos leads to zebrafish edema, complete loss of slit diaphragm, complete foot process effacement and increase in glomerular permeability. The phenotype can be rescued by expression of human protein 4.1O AA 506-553, the nephrin binding domain. AA 506-553 contain a MAPK and DEF (SFK phosphorylation) site. Inhibition of SFK and p38 attenuate significantly nephrin protein 4.1O interaction. 4.1O and nck compete for the binding to nephrin. Mutations of protein 4.1O at the DEF site reduces significantly nephrin protein 4.1O interaction. Protein 4.1O expression is increased in human DN, interestingly is reduced in streptozotocin treated mice.

Conclusion

Protein 4.1O is a novel podocyte protein that interacts with nephrin, GLEPP1, IQGAP1, Neph1 and the actin cytoskeleton. Protein 4.1O is essential for the intact glomerular filtration barrier. Knockdown of moe in zebrafish leads to nephrotic phenotype that is rescued by expression of human protein 4.1O AA 506-553. Nephrin protein 4.1O binding is regulated by SFKs and MAPK. The expression of protein 4.1O is increased in human DN and reduced in a mouse model.