RESUMEN
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.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Acuaporina 2/metabolismo , Membrana Celular/metabolismo , Túbulos Renales Colectores/citología , Antígenos de Histocompatibilidad Menor/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Proteína de Unión al GTP rhoA/metabolismo , Membrana Celular/efectos de los fármacos , Células HEK293 , Humanos , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadAsunto(s)
Proteínas de Anclaje a la Quinasa A/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Piridinas/química , Proteínas de Anclaje a la Quinasa A/metabolismo , Sitios de Unión , Unión Competitiva , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células HEK293 , Humanos , Simulación del Acoplamiento Molecular , Dominios y Motivos de Interacción de Proteínas , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/metabolismo , Estructura Terciaria de Proteína , Piridinas/síntesis química , Piridinas/metabolismoRESUMEN
In the principal cells of the renal collecting duct, arginine vasopressin (AVP) stimulates the synthesis of cAMP, leading to signaling events that culminate in the phosphorylation of aquaporin-2 water channels and their redistribution from intracellular domains to the plasma membrane via vesicular trafficking. The molecular mechanisms that control aquaporin-2 trafficking and the consequent water reabsorption, however, are not completely understood. Here, we used a cell-based assay and automated immunofluorescence microscopy to screen 17,700 small molecules for inhibitors of the cAMP-dependent redistribution of aquaporin-2. This approach identified 17 inhibitors, including 4-acetyldiphyllin, a selective blocker of vacuolar H(+)-ATPase that increases the pH of intracellular vesicles and causes accumulation of aquaporin-2 in the Golgi compartment. Although 4-acetyldiphyllin did not inhibit forskolin-induced increases in cAMP formation and downstream activation of protein kinase A (PKA), it did prevent cAMP/PKA-dependent phosphorylation at serine 256 of aquaporin-2, which triggers the redistribution to the plasma membrane. It did not, however, prevent cAMP-induced changes to the phosphorylation status at serines 261 or 269. Last, we identified the fungicide fluconazole as an inhibitor of cAMP-mediated redistribution of aquaporin-2, but its target in this pathway remains unknown. In conclusion, our screening approach provides a method to begin dissecting molecular mechanisms underlying AVP-mediated water reabsorption, evidenced by our identification of 4-acetyldiphyllin as a modulator of aquaporin-2 trafficking.
