Role and identification of protein kinase A anchoring proteins in vasopressin-mediated aquaporin-2 translocation  Enno Klussmann, Walter Rosenthal  Kidney International  Volume 60, Issue 2, Pages 446-449 (August 2001) DOI: 10.1046/j.1523-1755.2001.060002446.x Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 1 Detection of protein kinase A anchoring proteins (AKAPs) in soluble (S) and particulate (P) fractions derived from primary cultured rat renal inner medullary collecting ducts (IMCDs). The cells were cultured as described by Maric, Oksche, and Rosenthal and homogenized in a glass/teflon homogenizer in phosphate-buffered saline20. Nuclei and cell debris were removed by centrifugation at 1000 × g for 10 minutes. The homogenates were centrifuged at 100,000 × g for one hour to obtain particulate (pellet) and soluble (supernatant) fractions. Proteins (30 μg/lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 12%) and blotted onto nitrocellulose membranes. Regulatory type II subunits were radioactively labeled by incubation with [γ-32P]ATP and catalytic protein kinase A (PKA) subunits and added to the filter-bound proteins21. The experiment shown in the left panel was carried out in the presence of the control peptide Ht31-P (10 μmol/L). The experiment shown in the right panel was carried out in the presence of the peptide Ht31 (10 μmol/L), which inhibits the binding of regulatory PKA subunits to AKAPs. Signals were detected by exposure of the filters to phosphoimager plates and subsequent scanning of the plates in a phosphoimager (STORM820; Molecular Dynamics, Sunnyvale, CA, USA). Kidney International 2001 60, 446-449DOI: (10.1046/j.1523-1755.2001.060002446.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 2 A model for the involvement of AKAPs in the vasopressin/cAMP-triggered translocation of aquaporin 2 (AQP2) into the apical cell membranes of a renal collecting duct principal cell. Vasopressin binds to heptahelical vasopressin V2 receptors (V2R)16 located on the basolateral surface of principal cells. This initiates the synthesis of cAMP via the Gs/adenylyl cyclase (AC) system. cAMP in turn stimulates protein kinase A (PKA), which is targeted to various subcellular compartments by PKA anchoring proteins (AKAPs). Biochemical studies suggest that different AKAPs are specific for different compartments17,22,24. The inactive PKA holoenzyme consists of a dimer of regulatory (R) subunits (subtype RI or RII) to each of which a catalytic subunit (C) is bound. The binding to AKAPs is mediated by the dimer of R subunits. Two molecules of cAMP bind to each of the R subunits thereby lowering their affinity to the C subunits, which results in dissociation of C subunits from the holoenzyme/AKAP complex. The monomeric catalytic subunits (cPKA) phosphorylate substrates in their vicinity, including AQP2. Recently it has been demonstrated that not only the phosphorylation of AQP2 at Ser 256 by PKA is a prerequisite for its translocation, but also the anchoring of PKA to AKAPs, although the AKAPs involved have not yet been identified22,24. Kidney International 2001 60, 446-449DOI: (10.1046/j.1523-1755.2001.060002446.x) Copyright © 2001 International Society of Nephrology Terms and Conditions