Functional Characterization of Spinocerebellar Ataxia Associated Dynorphin A Mutant Peptides

Abstract

Mutations in the prodynorphin gene (PDYN) are associated with the development of spinocerebellar ataxia type 23 (SCA23). Pathogenic missense mutations are localized predominantly in the PDYN region coding for the dynorphin A (DynA) neuropeptide and lead to persistently elevated mutant peptide levels with neurotoxic properties. The main DynA target in the central nervous system is the kappa opioid receptor (KOR), a member of the G-protein coupled receptor family, which can elicit signaling cascades mediated by G-protein dissociation as well as β-arrestin recruitment. To date, a thorough analysis of the functional profile for the pathogenic SCA23 DynA mutants at KOR is still missing. To elucidate the role of DynA mutants, we used a combination of assays to investigate the differential activation of G-protein subunits and β-arrestin. In addition, we applied molecular modelling techniques to provide a rationale for the underlying mechanism. Our results demonstrate that DynA mutations, associated with a severe ataxic phenotype, decrease potency of KOR activation, both for G-protein dissociation as well as β-arrestin recruitment. Molecular modelling suggests that this loss of function is due to disruption of critical interactions between DynA and the receptor. In conclusion, this study advances our understanding of KOR signal transduction upon DynA wild type or mutant peptide binding.

Keywords: G-protein; TRUPATH; biased agonism; dynorphin; functional selectivity; kappa opioid receptor; ligand-directed signaling; spinocerebellar ataxia; β-arrestin.

Figure 1

Dynorphin A (DynA)_1-17 and DynA_R6W peptides signal at human kappa opioid receptor (hKOR). Left: Dose response curve of [³⁵S]GTPγS binding to membranes (10 μg) from cells expressing hKOR. Right: Summary showing the magnitude of the change in EC₅₀ induced by DynA_1-17 and DynA_R6W peptides in each experiment. Data are shown as mean ± SEM from six independent experiments. Mann–Whitney test, p-values are indicated in the figure. CPM: counts per minute.

Figure 2

DynA_1-17 and DynA_R6W peptides transducerome at hKOR. (A–F) Left: Dose response curves of TRUPATH (BRET assay) in HEK293 cells transiently transfected with hKOR and the different Gα subunits. (A–F) Right: Summary showing the magnitude of the change in EC₅₀ induced by DynA_1-17 and DynA_R6W peptides for each Gα subunit. Data are mean ± SEM. Mann–Whitney test, p-values are indicated in the figure.

Figure 3

Signal bias investigation for DynA_1-17 (Black), DynA_R6W (green), DynA_L5S (blue), and DynA_R9C (red) peptides at hKOR. (A) Left: Dose response curve obtained from TRUPATH (BRET assay) measuring G-protein dissociation in HEK293 cells transiently transfected with hKOR and the Gα3 subunit. Right: Magnitude change in EC₅₀ induced by DynA_1-17 SCA23 mutant peptides for Gα3 subunit in comparison to mean same day DynA_WT control. (B) Left: Dose response curve obtain from PRESTO-Tango assay measuring β-arrestin recruitment in HEK293 cells transiently transfected with hKOR. Right: Magnitude change in EC₅₀ induced by DynA_1-17 and the SCA23 mutant peptides in comparison to mean same day control. Data are shown as mean ± SEM. Wilcoxon Signed Rank Test; p-values are indicated in the figure.

Figure 4

Molecular modelling of DynA_WT, DynA_R6W and DynA_L5S. DynA R6 forms an ionic interaction with E297 (A), which is disrupted in the R6W mutant (B). (C) Dyn L5 is involved in hydrophobic contacts with a range of non-polar KOR residues lining a lipophilic side pocket. Similarly, these interactions cannot be formed with the L5S mutant (D). * and ** indicate the position of V320 and F143, respectively.