Dimerization of cAMP phosphodiesterase-4 (PDE4) in living cells requires interfaces located in both the UCR1 and catalytic unit domains

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Item Type:	Article
Title:	Dimerization of cAMP phosphodiesterase-4 (PDE4) in living cells requires interfaces located in both the UCR1 and catalytic unit domains
Creators Name:	Bolger, G.B., Dunlop, A.J., Meng, D., Day, J.P., Klussmann, E., Baillie, G.S., Adams, D.R. and Houslay, M.D.
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 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 additionally 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, because 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.
Keywords:	PDE4, Phosphodiesterase, cAMP, Cyclic AMP, Dimerization, Dimerisation, Rolipram
Source:	Cellular Signalling
ISSN:	0898-6568
Publisher:	Elsevier
Volume:	27
Number:	4
Page Range:	756-769
Date:	April 2015
Additional Information:	Copyright © 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Official Publication:	https://doi.org/10.1016/j.cellsig.2014.12.009
External Fulltext:	View full text on PubMed Central
PubMed:	View item in PubMed

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