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This Article Full Text Full Text (PDF) All Versions of this Article: 297/5/C1157    most recent 00219.2009v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Alli, A. A. Articles by Gower, W. R. PubMed PubMed Citation Articles by Alli, A. A. Articles by Gower, W. R., Jr.

Receptors and Signal Transduction

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

¹Research Service, James A. Haley Veterans Hospital; ²Department of Molecular Medicine, University of South Florida College of Medicine; and ³Cardiac 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 (PLC1) concurrently with AHNAK in nonneuronal cells. The recruitment of AHNAK from the nucleus is required for it to activate PLC1 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