The C Type Natriuretic Peptide Receptor Tethers AHNAK1 at the Plasma Membrane to Potentiate Arachidonic Acid-Induced Calcium Mobilization
3T3-L1, smooth muscle cells, rat gastric muscosa cell 1, phospholipase C
Digital Object Identifier (DOI)
Arachidonic acid (AA) liberated from membrane phospholipids is known to activate phospholipase C γ1 (PLCγ1) concurrently with AHNAK in nonneuronal cells. The recruitment of AHNAK from the nucleus is required for it to activate PLCγ1 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.
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Citation / Publisher Attribution
American Journal of Physiology-Cell Physiology, v. 297, issue 5, p. C1157-C1167
Scholar Commons Citation
Alli, Abdel A. and Gower, William R. Jr., "The C Type Natriuretic Peptide Receptor Tethers AHNAK1 at the Plasma Membrane to Potentiate Arachidonic Acid-Induced Calcium Mobilization" (2009). Molecular Medicine Faculty Publications. 85.