title: Dimerization of cAMP phosphodiesterase-4 (PDE4) in living cells requires interfaces located in both the UCR1 and catalytic unit domains.
abstract: PDE4 family cAMP phosphodiesterases play a pivotal role in determining compartmentalised cAMP signalling through targeted cAMP breakdown. Expressing the widely found PDE4D5 isoform, as both bait and prey in a yeast 2-hybrid system, we demonstrated interaction consistent with the notion that long PDE4 isoforms form dimers. Four potential dimerization sites were uncovered using a scanning peptide array approach, where a recombinant purified PDE4D5 fusion protein was used to probe a 25-mer library of overlapping peptides covering the entire PDE4D5 sequence. Key residues involved in PDE4D5 dimerization were defined using a site-directed mutagenesis programme directed by an alanine scanning peptide array approach. Critical residues stabilising PDE4D5 dimerization were defined within the regulatory UCR1 region found in long, but not short, PDE4 isoforms, namely the Arg(173), Asn(174) and Asn(175) (DD1) cluster. Disruption of the DD1 cluster was not sufficient, in itself, to destabilise PDE4D5 homodimers. Instead, disruption of an additional interface, located on the PDE4 catalytic unit, was also required to convert PDE4D5 into a monomeric form. This second dimerization site on the conserved PDE4 catalytic unit is dependent upon a critical ion pair interaction. This involves Asp(463) and Arg(499) in PDE4D5, which interact in a trans fashion involving the two PDE4D5 molecules participating in the homodimer. PDE4 long isoforms adopt a dimeric state in living cells that is underpinned by two key contributory interactions, one involving the UCR modules and one involving an interface on the core catalytic domain. We propose that short forms do not adopt a dimeric configuration because, in the absence of the UCR1 module, residual engagement of the remaining core catalytic domain interface provides insufficient free energy to drive dimerization. The functioning of PDE4 long and short forms is thus poised to be inherently distinct due to this difference in quaternary structure.
authors.affiliation: Departments of Medicine and Pharmacology, University of Alabama, Birmingham, AL 35294, USA.
authors.forename: Graeme B
authors.initials: GB
authors.lastname: Bolger
authors.affiliation: Institute of Cardiovascular and Medical Science, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.
authors.forename: Allan J
authors.initials: AJ
authors.lastname: Dunlop
authors.affiliation: Institute of Cardiovascular and Medical Science, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.
authors.forename: Dong
authors.initials: D
authors.lastname: Meng
authors.affiliation: Institute of Cardiovascular and Medical Science, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.
authors.forename: Jon P
authors.initials: JP
authors.lastname: Day
authors.affiliation: Max Delbrueck Center for Molecular Medicine, German Centre for Cardiovascular Research (DZHK), Berlin, Germany.
authors.forename: Enno
authors.initials: E
authors.lastname: Klussmann
authors.affiliation: Institute of Cardiovascular and Medical Science, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.
authors.forename: George S
authors.initials: GS
authors.lastname: Baillie
authors.affiliation: Institute of Chemical Sciences, Heriot-Watt University, Riccarton, Edinburgh EH14 4AS, Scotland, United Kingdom.
authors.forename: David R
authors.initials: DR
authors.lastname: Adams
authors.affiliation: Institute of Pharmaceutical Sciences, King's College London, London SE1 9NH, United Kingdom. Electronic address: miles.houslay@kcl.ac.uk.
authors.forename: Miles D
authors.initials: MD
authors.lastname: Houslay
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D000242:Cyclic AMP / Q000378:metabolism; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary
chemical_list: D010455:Peptides; D000242:Cyclic AMP; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; C517376:PDE4D protein, human
keywords: Dimerisation; Dimerization; PDE4; Phosphodiesterase; Rolipram; cAMP; cyclic AMP