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Vol. 18, Issue 7, 2768-2777, July 2007

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Binding of CAP70 to Inducible Nitric Oxide Synthase and Implications for the Vectorial Release of Nitric Oxide in Polarized Cells

Inmaculada Navarro-Lérida^*, Mónica Martínez-Moreno^*, Iván Ventoso, Alberto Álvarez-Barrientos, and Ignacio Rodríguez-Crespo^*

*Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, 28040 Madrid, Spain; Centro de Biología Molecular "Severo Ochoa," Consejo Superior de Investigaciones Científicas-Universidad Autónoma, Facultad de Ciencias, Cantoblanco, Universidad Autónoma de Madrid, 28049 Madrid, Spain; and Fundación Centro Nacional de Investigaciones Cardiovasculares, 28029 Madrid, Spain

Submitted December 13, 2006; Revised April 19, 2007; Accepted May 3, 2007
Monitoring Editor: Asma Nusrat

In this article we analyze the mechanisms by which the C-terminal four amino acids of inducible nitric oxide synthase (NOS2) interact with proteins that contain PDZ (PSD-95/DLG/ZO-1) domains resulting in the translocation of NOS2 to the cellular apical domain. It has been reported that human hepatic NOS2 associates to EBP50, a protein with two PDZ domains present in epithelial cells. We describe herein that NOS2 binds through its four carboxy-terminal residues to CAP70, a protein that contains four PDZ modules that is targeted to apical membranes. Interestingly, this interaction augments both the cytochrome c reductase and ·NO-synthase activities of NOS2. Binding of CAP70 to NOS2 also results in an increase in the population of active NOS2 dimers. In addition, CAP70 participates in the correct subcellular targeting of NOS2 in a process that is also dependent on the acylation state of the N-terminal end of NOS2. Hence, nonpalmitoylated NOS2 is unable to progress toward the apical side of the cell despite its interaction with either EBP50 or CAP70. Likewise, if we abrogate the interaction of NOS2 with either EBP50 or CAP70 by fusing the GFP reporter to the carboxy-terminal end of NOS2 palmitoylation is not sufficient to confer an apical targeting.

Address correspondence to: Ignacio Rodríguez-Crespo (nacho{at}bbm1.ucm.es)

Abbreviations used: CAP70, CFTR-associated protein of 70 kDa; CFTR, cystic fibrosis transmembrane conductance; EBP50, ezrin-radixin-moesin-binding phosphoprotein of 50 kDa; GFP, green fluorescent protein; hNOS2, human hepatic NOS2; mNOS2, mouse macrophage NOS2; NOS, nitric oxide synthase; NOS2, inducible nitric oxide synthase; PDZ, PSD-95/DLG/ZO-1.

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