Abstract 17934: Phosphoprotein Profiling Unravels Novel Phosphoproteins That Distinguish Intracellular cGMP Signaling by Natriuretic Peptide- and Nitric Oxide-Activated Guanylyl Cyclases
Intracellular cGMP regulates vascular smooth muscle cell (VSMC) growth and proliferation. Cellular guanylyl cyclases (GCs) are generally divided into two groups: natriuretic peptide- activated-GCs (NPGCs) and nitric oxide-activated GCs (NOGCs). NPGCs are homodimeric, membrane-associated, receptors for atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). NOGCs are heme-containing heterodimers. Whether signaling pathways for cGMP formed by these two forms of GC is the same is not known. In order to begin to address this, we screened for the abundance of approximately 200 different phosphoproteins in cultured human vascular smooth muscle cells treated with 1) ANP (10nM for 1 hr) and 2) NO donor S-Nitroso Penicillamine (SNAP) (100uM for 1hr). Intracellular cGMP content was the same with both treatments (ANP 27 pmol/mg protein vs. SNAP 29 pmol/mg protein P<0.05). Phosphoproteins were separated and detected using 2-dimensional Difference in Gel Electrophoresis (2D-DIGE) with pro-Q diamond staining. Spot volumes (an index of phosphoprotein abundance) were compared between the two treatments. At least 25 phosphoproteins with greater than 3 — fold difference in abundance between the two treatment groups were detected. Selected phosphoproteins from this group were identified using Matrix assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS/MS) and Mascot analysis. Among those more abundant in cells treated with SNAP versus ANP were the cytoskeletal proteins vimentin, spectrin, annexin-A2, and cytoplasmic protein nipsnap3A. Those more abundant in cells treated with ANP versus SNAP included mitochondria- and endoplasmic reticulum-associated proteins hexokinase-1, pitrilysin metallopeptidase-1, aldehyde dehydrogenase, and prolyl-4-hydroxylase beta. These results provide evidence that signaling pathways and phsophorylation targets for intracellular cGMP generated by NOGCs and NPGCs are different in human vascular smooth muscle cells.
- © 2010 by American Heart Association, Inc.