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Am J Physiol Cell Physiol. 2004 May;286(5):C1037-44. Epub 2003 Dec 18.

Role of a PDZ1 domain of NHERF1 in the binding of airway epithelial RACK1 to NHERF1.

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Pediatric Pulmonology, Case Western Reserve Univ., BRB, Rm. 824, 2109 Adelbert Rd., Cleveland, OH 44106-4948, USA.


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.

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