The C type natriuretic peptide receptor tethers AHNAK1 at the plasma membrane to potentiate arachidonic acid-induced calcium mobilization

1 Research Service, James A. Haley Veterans Hospital; 2 Department of Molecular Medicine, University of South Florida College of Medicine; and 3 Cardiac Hormone Center, University of South Florida, Tampa, Florida Submitted 18 May 2009 ; accepted in final form 20 August 2009 Arachidonic acid (AA) liberated from membrane phospholipids is known to activate phospholipase C 1 (PLC 1) concurrently with AHNAK in nonneuronal cells. The recruitment of AHNAK from the nucleus is required for it to activate PLC 1 at the plasma membrane. Here, we identify the C-type natriuretic peptide receptor (NPR-C), an atypical G protein-coupled receptor, as a protein binding partner for AHNAK1 in various cell types. Mass spectrometry and MASCOT analysis of excised bands from NPR-C immunoprecipitation studies revealed multiple signature peptides corresponding to AHNAK1. Glutathione S -transferase (GST) pulldown assays using GST- AHNAK1 fusion proteins corresponding to each of the distinct domains of AHNAK1 showed the C1 domain of AHNAK1 associates with NPR-C. The role of NPR-C in mediating AA-dependent AHNAK1 calcium signaling was explored in various cell types, including 3T3-L1 preadipocytes during the early stages of differentiation. Sucrose density gradient centrifugation studies showed AHNAK1 resides in the nucleus, cytoplasm, and at the plasma membrane, but small interfering RNA (siRNA)-mediated knockdown of NPR-C resulted in AHNAK1 accumulation in the nucleus. Overexpression of a portion of AHNAK1 resulted in augmentation of intracellular calcium mobilization, whereas siRNA-mediated knockdown of NPR-C or AHNAK1 protein resulted in attenuation of intracellular calcium mobilization in response to phorbol 12-myristate 13-acetate. We characterize the novel association between AHNAK1 and NPR-C and provide evidence that this association potentiates the AA-induced mobilization of intracellular calcium. We address the role of intracellular calcium in the various cell types that AHNAK1 and NPR-C were found to associate. 3T3-L1; smooth muscle cells; rat gastric muscosa cell 1; phospholipase C Address for reprint requests and other correspondence: W. R. Gower, Jr., James A. Haley VA Hospital, Research Service (151), 13000 Bruce B. Downs Blvd., Tampa, FL 33612 (e-mail: wgower{at}health.usf.edu ).