Role of a PDZ1 domain of NHERF1 in the binding of airway epithelial RACK1 to NHERF1.
In past studies, we demonstrated regulation of CFTR Cl channel function by protein kinase C (PKC)-epsilon through the binding of PKC-epsilon to RACK1 (a receptor for activated C-kinase) and of RACK1 to human Na(+)/H(+) exchanger regulatory factor (NHERF1). In this study, we investigated the site of RACK1 binding on NHERF1 using solid-phase and solution binding assays and pulldown, immunoprecipitation, and (36)Cl efflux experiments. Recombinant RACK1 binding to glutathione S-transferase (GST)-tagged PDZ1 domain of NHERF1 was 10-fold higher than its binding to GST-tagged PDZ2 domain of NHERF1. PDZ1 binds to RACK1 in a dose-dependent manner and vice versa, with similar binding constants of 1.67 and 1.26 microg, respectively. Interaction of the PDZ1 domain with RACK1 was not blocked by binding of activated PKC-epsilon to RACK1. A GST-tagged PDZ1 domain pulled down endogenous RACK1 from Calu-3 cell lysate. An internal 11-amino acid motif embedding the GYGF carboxylate binding loop of PDZ1 binds to RACK1, inhibits binding of recombinant NHERF1 and RACK1, pulls down endogenous RACK1 from Calu-3 cell lysate, and blocks coimmunoprecipitation of endogenous RACK1 with endogenous NHERF1 but does not affect cAMP-dependent activation of CFTR. A similar amino acid sequence in the PDZ2 domain did not bind RACK1. Our results indicate binding of Calu-3 RACK1 predominantly to the PDZ1 domain of NHERF1 at a site encompassing the GYGF loop of the PDZ1 domain and a site on RACK1 distinct from a PKC-epsilon binding site. CFTR activation by cAMP-generating agent is not affected by loss of RACK1-NHERF1 interaction.
Liedtke
Carole M
CM
Pediatric Pulmonology, Case Western Reserve Univ., BRB, Rm. 824, 2109 Adelbert Rd., Cleveland, OH 44106-4948, USA. carole.liedtke@case.edu
Raghuram
Viswanathan
V
Yun
C Chris
CC
Wang
Xiangyun
X
D020816:Amino Acid Motifs / Q000502:physiology; D002460:Cell Line; D006801:Humans; D010750:Phosphoproteins / Q000737:chemistry* / Q000502:physiology*; D011493:Protein Kinase C / Q000378:metabolism; D051744:Protein Kinase C-epsilon; D017434:Protein Structure, Tertiary / Q000502:physiology; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020545:Respiratory Mucosa / Q000166:cytology / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers
D010750:Phosphoproteins; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor; C497255:PRKCE protein, human; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon