Role of PSD95 in membrane association and catalytic activity of nNOSalpha in nitrergic varicosities in mice gut.
We have recently shown that membrane association of neuronal nitric oxide synthase-alpha (nNOSalpha) is critical in the regulation of synthesis of NO during nitrergic neurotransmission. The purpose of this study was to examine the role of the synapse-associated proteins (SAPs) in membrane association of nNOSalpha. Varicosities (swellings on terminal axons) were isolated from mice gastrointestinal tract and examined for nNOSalpha, postsynaptic density protein 95 (PSD95), and membrane interactions by coimmunoprecipitation and SDS-PAGE. Our results show that PSD95 protein was present in the membrane fraction of the nerve varicosity, whereas both PSD95 and SAP97 were present in the cytosol. nNOSalpha was associated with PSD95 but not SAP97. nNOSalpha-PSD95 complex was bound to the membrane via palmitoylation of PSD95. Depalmitoylation of PSD95 with 2-bromopalmitate dislocates nNOSalpha and PSD95 from the varicosity membrane and abolishes NO production. These studies show that palmitoylation of PSD95 anchors nNOSalpha to the varicosity membrane and that it is obligatory for NO production by the enzyme. Palmitoylation of PSD95 may provide a novel target for regulation of nitrergic neurotransmission.
Chaudhury
Arun
A
Gastrointestinal Division, Veterans Affairs Boston Healthcare System and Harvard Medical School, Center for Swallowing and Motility Disorders, Boston, Massachusetts, USA.
He
Xue-Dao
XD
Goyal
Raj K
RK
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology*; D003600:Cytosol / Q000378:metabolism; D000074288:Discs Large Homolog 1 Protein; D000076122:Disks Large Homolog 4 Protein; D004591:Electrophoresis, Polyacrylamide Gel; D017615:Enteric Nervous System / Q000187:drug effects / Q000201:enzymology*; D004789:Enzyme Activation; D041981:Gastrointestinal Tract / Q000294:innervation*; D051528:Guanylate Kinases; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D054878:Lipoylation; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D026602:Nitrergic Neurons / Q000187:drug effects / Q000201:enzymology*; D009569:Nitric Oxide / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I / Q000378:metabolism*; D010168:Palmitates / Q000494:pharmacology; D017729:Presynaptic Terminals / Q000187:drug effects / Q000201:enzymology*; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D009435:Synaptic Transmission
D048868:Adaptor Proteins, Signal Transducing; D000074288:Discs Large Homolog 1 Protein; D000076122:Disks Large Homolog 4 Protein; C000614189:Dlg1 protein, mouse; C501447:Dlg4 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D010168:Palmitates; C022776:2-bromopalmitate; D009569:Nitric Oxide; D052248:Nitric Oxide Synthase Type I; C496321:Nos1 protein, mouse; D051528:Guanylate Kinases
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