The C type natriuretic peptide receptor tethers AHNAK1 at the plasma membrane to potentiate arachidonic acid-induced calcium mobilization.
Arachidonic acid (AA) liberated from membrane phospholipids is known to activate phospholipase C gamma1 (PLCgamma1) concurrently with AHNAK in nonneuronal cells. The recruitment of AHNAK from the nucleus is required for it to activate PLCgamma1 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.
Alli
Abdel A
AA
Research Service, James A. Haley Veterans Hospital, Tampa, Florida 33612, USA.
Gower
William R
WR
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D015153:Blotting, Western; D020013:Calcium Signaling / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal; D009131:Muscle, Smooth, Vascular / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism*; D051381:Rats; D017461:Receptors, Atrial Natriuretic Factor / Q000378:metabolism*
C075176:AHNAK protein, human; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D016718:Arachidonic Acid; D017461:Receptors, Atrial Natriuretic Factor; C073616:atrial natriuretic factor receptor C