Background Alterations in cellular calcium homeostasis have been recognised as one of several pathogenic mechanisms in Huntington's disease (HD). The discovery of a direct interaction between the C terminal extremity (C-ter) of inositol-3-phosphate receptor (IP3R) and huntingtin (htt) associated protein 1 (HAP1) has shed light on the function of HAP1 in calcium homeostasis regulation. The C-ter of IP3R consists of a small domain in continuation of the transmembrane region; it has important regulatory functions on the IP3R.
Aim We wondered whether this regulatory domain is present in other membrane proteins, which might therefore interact with HAP1 and Htt, and could display altered functions in the presence of mutant htt. We searched for Cter-IP3R homologous sequences and discovered that the C-ter domain of another transmembrane channel—the Ryanodine Receptor (RYR), which is also a key regulator of calcium homeostasis—displays a striking sequence homology with C-ter-IP3R.
Methods/techniques GST-pull down, Co-IP, immunocytochemistry, FLIM-FRET.
Results In GST-pull down experiments, a specific interaction of HAP1 with C-ter-RYR and C-ter-IP3R, respectively, was observed. Mutant and wt Htt was pulled down in co-immunoprecipitation experiments using four different antibodies against RYR. Transiently expressed GFP tagged C-ter-RYR together with mRFP labelled N-ter-htt colocalised in N2a cells whereas expression of C-ter-RYR with mRFP alone did not. Immunocytochemistry of primary neurons showed a colocalisation of endogenous htt and RYR. To further assess the interaction of htt and RYR in primary neurons we performed FLIM-FRET based assays in a 96 well format using a high resolution fluorescent plate reader. Close proximity of RYR and htt was detected between the two proteins, as indicated by a significant shortening in lifetime of the donor fluorochrome compared with the negative control in which the acceptor fluorophore was not present. Significant lifetime shortening of the donor fluorochrome was observed independently of RYR or htt used as donor molecule. In summary, these results clearly suggest an interaction of RYR with htt and HAP1. Further work is in progress to clarify the effect of mutant htt on the channel activity of the ryanodine receptor.
- ryanodine receptor
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Funding Department of Neurology, Ulm University. Unrestricted research grant to GBL, INSERM, CNRS, Universite de Strasbourg (UDS), EUROSCA, Association Huntington France, ANR. National Institute of Health Grant AG13617 to ABY.