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Am J Physiol Cell Physiol (December 18, 2003). doi:10.1152/ajpcell.00222.2003
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Submitted on May 30, 2003
Accepted on December 10, 2003

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

Carole M Liedtke1*, Viswanathan Raghuram2, Chris Yun3, and Xiangyun Wang1

1 Pediatrics, Case Western Reserve University, Cleveland, OH, USA
2 Physiology, University of Pennsylvania, Philadelphia, PA, USA
3 Medicine, Emory University, Atlanta, GA, USA

* To whom correspondence should be addressed. E-mail: CXL7{at}CWRU.EDU.

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 36Cl efflux experiments. Recombinant RACK1 binding to 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 constant of 1.67 µg and 1.26 µg. Interaction of the PDZ1 domain to 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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