An AKAP-Lbc-RhoA interaction inhibitor promotes the translocation of aquaporin-2 to the plasma membrane of renal collecting duct principal cells

Stimulation of renal collecting duct principal cells with antidiuretic hormone (arginine-vasopressin, AVP) results in inhibition of the small GTPase RhoA and the enrichment of the water channel aquaporin-2 (AQP2) in the plasma membrane. The membrane insertion facilitates water reabsorption from primary urine and fine-tuning of body water homeostasis. Rho guanine nucleotide exchange factors (GEFs) interact with RhoA, catalyze the exchange of GDP for GTP and thereby activate the GTPase. However, GEFs involved in the control of AQP2 in renal principal cells are unknown. The A-kinase anchoring protein, AKAP-Lbc, possesses GEF activity, specifically activates RhoA, and is expressed in primary renal inner medullary collecting duct principal (IMCD) cells. Through screening of 18,431 small molecules and synthesis of a focused library around one of the hits, we identified an inhibitor of the interaction of AKAP-Lbc and RhoA. This molecule, Scaff10-8, bound to RhoA, inhibited the AKAP-Lbc-mediated RhoA activation but did not interfere with RhoA activation through other GEFs or activities of other members of the Rho family of small GTPases, Rac1 and Cdc42. Scaff10-8 promoted the redistribution of AQP2 from intracellular vesicles to the periphery of IMCD cells. Thus, our data demonstrate an involvement of AKAP-Lbc-mediated RhoA activation in the control of AQP2 trafficking.

Six (biotinylation) to 13 (Rhotekin assay) days after seeding, cells were used for experiments. 24 h before starting the experiment, cells were incubated in medium without DBcAMP and nystatin in order to increase the perinuclear localization of AQP2.

Plasmids
The AKAP-Lbc/DHPH sequence fused to an N-terminal GFP-tag and to C-terminal His 6 -tag was subcloned into the pTT5 plasmid (ampicillin resistance) containing a secretion sequence derived from an immunoglobulin (human IgG 1 ). [10] The full-length sequence refers to a secretion sequence derived from an immunoglobulin (bp 1-56 of accession number

Luciferase reporter assays
Luciferase reporter assays were carried out according to the manufacture's protocol "Dualluciferase reporter assay" (Promega). In brief, HEK293-Zellen were transiently transfected to express two luciferases and the indicated GEFs (Supplemental Figure 1). The serum response element (SRE-) promotor controls the firefly luciferase. Active RhoA stimulates serum response factor (SRF), which in turn binds to the SRE and stimulates expression of firefly luciferase. 20 hours post transfection the cells were exposed to Scaf10 and another 4 hours later they were lysed in Passive Lysis Buffer (Promega). Luciferase activity, i.e.

1 H NMR
1 H NMR spectra were recorded on a Bruker AV 300 (1H: 300 MHz). Chemical shifts are depicted in parts per million (ppm). As internal standard, the measured NMR-spectra were normalised to the solvent peak used for recording the spectrum (dimethylsulfoxide-d 6 (DMSO): 1H: 2.50 ppm).

LC/MS
All mass spectra were recorded on a QTrap 4000 (Applied Biosystems) connected to a in vivo tests had a purity of >95%. Absorption maxima were extracted from spectra generated by the DAD detector during LC/MS analyses.