title: Role of Ser102 and Ser104 as regulators of cGMP hydrolysis by PDE5A.
abstract: BACKGROUND
  Phosphodiesterases (PDEs) cleave phosphodiester bonds in cyclic nucleotides and play diverse roles in cell biology. PDE5A is a cytoplasmic phosphodiesterase which specifically degrades cyclic guanosine monophosphate (cGMP), a cell signaling molecule that plays important roles in neuronal signaling and vascular smooth muscle contraction. Inhibition of PDE5A induces headache resembling migraine headaches.
  
  
  OBJECTIVE
  To test the hypothesis that Ser102 and Ser104 in PDE5A and/or their phosphoserine derivatives 1) regulate the intracellular localization and/or activity of PDE5A, and 2) modulate the interaction between PDE5A and pharmaceutical reagents in clinical or pre-clinical use for migraine headaches and other types of vascular dysfunction.
  
  
  METHODS
  Wild type PDE5A or PDE5A with substitution mutations (Ser102Ala, Ser104Ala or Ser102Ala/Ser104Ala) were overexpressed in SK-N-AS neuroblastoma cells as C-terminal fusions with green fluorescent protein. Transfected cells were treated with sildenafil, cilostazol, glyceryl trinitrate, calcitonin gene-related peptide (CGRP) or sumatriptan. PDE5A-GFP fusion proteins were localized in fixed cells by immunofluorescence and PDE activity was quantified in cell extracts by standard in vitro assay using [3H] cGMP.
  
  
  RESULTS
  The intracellular distribution of wild-type, single and double mutant PDE5A was similar and was not altered by exposure to sildenafil, cilostazol, glyceryl trinitrate, calcitonin gene-related peptide (CGRP) or sumatriptan. PDE5 activity was similar for wild type, Ser102Ala and Ser104Ala PDE5A, but activity of the Ser102Ala/Ser104Ala mutant was approximately two-fold higher than wild type. Double mutant Ser102Ala/Ser104Ala migrated as a single band on a native acrylamide gel, while wild-type and single mutant PDE5A migrated as a doublet.
  
  
  CONCLUSIONS
  Ser102 and Ser104 may influence the conformational flexibility of PDE5A, which may in turn influence phosphorylation status, allosteric regulation by cGMP or other as yet unknown regulatory mechanisms for PDE5A. PDE5A activation could be important in reversal of migraine-like headache.
authors.affiliation: Lundbeck Foundation Center for Neurovascular signaling (LUCENS), Glostrup Research Institute, Glostrup Hospital, University of Copenhagen, Glostrup, Denmark.
authors.forename: Julie
authors.initials: J
authors.lastname: Carøe Nordgaard
authors.affiliation: Department of Clinical Experimental Research, Glostrup Hospital, University of Copenhagen, Glostrup, Denmark.
authors.forename: Lars Schack
authors.initials: LS
authors.lastname: Kruse
authors.affiliation: Department of Diagnostics, Clinical Biochemistry section Glostrup Hospital, University of Copenhagen, Glostrup, Denmark.
authors.forename: Steen
authors.initials: S
authors.lastname: Gammeltoft
authors.affiliation: Lundbeck Foundation Center for Neurovascular signaling (LUCENS), Glostrup Research Institute, Glostrup Hospital, University of Copenhagen, Glostrup, Denmark; Department of Neurology, Neurovascular Research Unit, Herlev Hospital, University of Copenhagen, Herlev, Denmark.
authors.forename: Christina Rostrup
authors.initials: CR
authors.lastname: Kruuse
mesh_terms: D019943:Amino Acid Substitution; D015740:Calcitonin Gene-Related Peptide / Q000494:pharmacology; D020134:Catalytic Domain; D002460:Cell Line; D000077407:Cilostazol; D006152:Cyclic GMP / Q000378:metabolism*; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008958:Models, Molecular; D005996:Nitroglycerin / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010879:Piperazines / Q000494:pharmacology; D011687:Purines / Q000494:pharmacology; D012694:Serine / Q000378:metabolism*; D000068677:Sildenafil Citrate; D013449:Sulfonamides / Q000494:pharmacology; D018170:Sumatriptan / Q000494:pharmacology; D013777:Tetrazoles / Q000494:pharmacology
chemical_list: D010726:Phosphodiesterase Inhibitors; D010879:Piperazines; D011687:Purines; D013449:Sulfonamides; D013777:Tetrazoles; D012694:Serine; D018170:Sumatriptan; D000068677:Sildenafil Citrate; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; C517381:PDE5A protein, human; D005996:Nitroglycerin; D006152:Cyclic GMP; D015740:Calcitonin Gene-Related Peptide; D000077407:Cilostazol