Smith, K. J. et al. (2007) 1H NMR structural and functional characterisation of a cAMP-specific phosphodiesterase-4D5 (PDE4D5) N-terminal region peptide that disrupts PDE4D5 interaction with the signalling scaffold proteins, βarrestin and RACK1. Cellular Signalling, 19(12), pp. 2612-2624. (doi: 10.1016/j.cellsig.2007.08.015)
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Publisher's URL: http://dx.doi.org/10.1016/j.cellsig.2007.08.015
Abstract
The unique 88 amino acid N-terminal region of cAMP-specific phosphodiesterase-4D5 (PDE4D5) contains overlapping binding sites conferring interaction with the signaling scaffold proteins, βarrestin and RACK1. A 38-mer peptide, whose sequence reflected residues 12 through 49 of PDE4D5, encompasses the entire N-terminal RACK1 Interaction Domain (RAID1) together with a portion of the βarrestin binding site. 1H NMR and CD analyses indicate that this region has propensity to form a helical structure. The leucine-rich hydrophobic grouping essential for RACK1 interaction forms a discrete hydrophobic ridge located along a single face of an amphipathic α-helix with Arg34 and Asn36, which also play important roles in RACK1 binding. The Asn22/Pro23/Trp24/Asn26 grouping, essential for RACK1 interaction, was located at the N-terminal head of the amphipathic helix that contained the hydrophobic ridge. RAID1 is thus provided by a distinct amphipathic helical structure. We suggest that the binding of PDE4D5 to the WD-repeat protein, RACK1, may occur in a manner akin to the helix–helix interaction shown for Gγ binding to the WD-repeat protein, Gβ. A more extensive section of the PDE4D5 N-terminal sequence (Thr11-Ala85) is involved in βarrestin binding. Several residues within the RAID1 helix contribute to this interaction however. We show here that these residues form a focused band around the centre of the RAID1 helix, generating a hydrophobic patch (from Leu29, Val30 and Leu33) flanked by polar/charged residues (Asn26, Glu27, Asp28, Arg34). The interaction with βarrestin exploits a greater circumference on the RAID1 helix, and involves two residues (Glu27, Asp28) that do not contribute to RACK1 binding. In contrast, the interaction of RACK1 with RAID1 is extended over a greater length of the helix and includes Leu37/Leu38, which do not contribute to βarrestin binding. A membrane-permeable, stearoylated Val12-Ser49 38-mer peptide disrupted the interaction of both βarrestin and RACK1 with endogenous PDE4D5 in HEKB2 cells, whilst a cognate peptide with a Glu27Ala substitution selectively failed to disrupt PDE4D5/RACK1 interaction. The stearoylated Val12-Ser49 38-mer peptide enhanced the isoprenaline-stimulated PKA phosphorylation of the β2-adrenergic receptors (β2AR) and its activation of ERK, whilst the Glu27Ala peptide was ineffective in both these regards.
| Item Type: | Articles |
|---|---|
| Status: | Published |
| Refereed: | Yes |
| Glasgow Author(s) Enlighten ID: | Houslay, Professor Miles and Baillie, Professor George and McCahill, Dr Angela and Dunlop, Dr Allan and Houslay, Mr Thomas |
| Authors: | Smith, K. J., Baillie, G. S., Hyde, E. I., Li, X., Houslay, T., McCahill, A., Dunlop, A. J., Bolger, G. B., Klussmann, E., Adams, D. R., and Houslay, M. D. |
| College/School: | College of Medical Veterinary and Life Sciences > School of Cardiovascular & Metabolic Health College of Medical Veterinary and Life Sciences > School of Psychology & Neuroscience |
| Journal Name: | Cellular Signalling |
| Publisher: | Elsevier Inc. |
| ISSN: | 0898-6568 |
| ISSN (Online): | 1873-3913 |
| Related URLs: |
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