PDZK1: II. an anchoring site for the PKA-binding protein D-AKAP2 in renal proximal tubular cells.
BACKGROUND
PDZK1, a multiple PDZ protein, was recently found to interact with the type IIa Na/Pi cotransporter (NaPi-IIa) in renal proximal tubular cells. In a preceding study, yeast two-hybrid screens using single PDZ domains of PDZK1 as baits were performed. Among the identified proteins, a C-terminal fragment of the dual-specific A-kinase anchoring protein 2 (D-AKAP2) was obtained by screening PDZ domain 4.


METHODS
After its molecular cloning by means of RACE, the renal expression of D-AKAP2 was analyzed by real-time polymerase chain reaction (PCR) and immunohistochemistry. Protein interactions were characterized by overlays, pull-downs, and immunoprecipitations from transfected opossum kidney (OK) cells.


RESULTS
Based on 5'-RACE and PDZK1 overlays of mouse kidney cortex separated by two-dimensional electrophoresis, it was suggested that the renal isoform of D-AKAP2 in mouse comprises 372 amino acids and exists as a protein of >40 kD. Immunohistochemistry and real-time PCR localized D-AKAP2 only to the subapical pole of proximal tubular cells in mouse kidney. In pull-down experiments, D-AKAP2 tightly bound PDZK1 as well as N+/H+ exchanger regulator factor (NHERF-1), but the latter with an apparent fourfold lower affinity. Similarly, His-tagged D-AKAP2 specifically retained PDZK1 from mouse kidney cortex homogenate. In addition, myc-tagged D-AKAP2 and HA-tagged PDZK1 co-immunoprecipitated from transfected OK cells.


CONCLUSIONS
We conclude that D-AKAP2 anchors protein kinase A (PKA) to PDZK1 and to a lesser extent to NHERF-1. Since PDZK1 and NHERF-1 both sequester NaPi-IIa to the apical membrane, D-AKAP2 may play an important role in the parathyroid hormone (PTH)-mediated regulation of NaPi-IIa by compartmentalization of PKA.
Gisler
Serge M
SM
Department of Physiology, University of Zürich, Zürich, Switzerland.
Madjdpour
Caveh
C
Bacic
Desa
D
Pribanic
Sandra
S
Taylor
Susan S
SS
Biber
Jürg
J
Murer
Heini
H
D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009865:Oocytes; D011485:Protein Binding / Q000502:physiology; D014982:Xenopus laevis
D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing; C108772:Akap10 protein, mouse; D002352:Carrier Proteins; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D017868:Cyclic AMP-Dependent Protein Kinases