Role of Ser102 and Ser104 as regulators of cGMP hydrolysis by PDE5A

PLoS One. 2014 Sep 23;9(9):e107627. doi: 10.1371/journal.pone.0107627. eCollection 2014.

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.

Aim: 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.

Interpretation: 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Calcitonin Gene-Related Peptide / pharmacology
  • Catalytic Domain
  • Cell Line
  • Cilostazol
  • Cyclic GMP / metabolism*
  • Cyclic Nucleotide Phosphodiesterases, Type 5 / chemistry
  • Cyclic Nucleotide Phosphodiesterases, Type 5 / genetics*
  • Cyclic Nucleotide Phosphodiesterases, Type 5 / metabolism*
  • Humans
  • In Vitro Techniques
  • Models, Molecular
  • Nitroglycerin / pharmacology
  • Phosphodiesterase Inhibitors / pharmacology
  • Piperazines / pharmacology
  • Purines / pharmacology
  • Serine / metabolism*
  • Sildenafil Citrate
  • Sulfonamides / pharmacology
  • Sumatriptan / pharmacology
  • Tetrazoles / pharmacology

Substances

  • Phosphodiesterase Inhibitors
  • Piperazines
  • Purines
  • Sulfonamides
  • Tetrazoles
  • Serine
  • Sumatriptan
  • Sildenafil Citrate
  • Cyclic Nucleotide Phosphodiesterases, Type 5
  • PDE5A protein, human
  • Nitroglycerin
  • Cyclic GMP
  • Calcitonin Gene-Related Peptide
  • Cilostazol

Grants and funding

LUCENS. A.P. Møller Foundation for the Advancement of Medical Science. Christian and Ottilia Brorsons travel grant. Cool Sorption Foundation of 1988. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.