Pentameric quaternary structure of the intracellular domain of serotonin type 3A receptors

Abstract

In spite of extensive efforts over decades an experimentally-derived structure of full-length eukaryotic pentameric ligand-gated ion channels (pLGICs) is still lacking. These pharmaceutically highly-relevant channels contain structurally well-conserved and characterized extracellular and transmembrane domains. The intracellular domain (ICD), however, has been orphaned in structural studies based on the consensus assumption of being largely disordered. In the present study, we demonstrate for the first time that the serotonin type 3A (5-HT3A) ICD assembles into stable pentamers in solution in the absence of the other two domains, thought to be the drivers for oligomerization. Additionally, the soluble 5-HT3A-ICD construct interacted with the protein RIC-3 (resistance to inhibitors of cholinesterase). The interaction provides evidence that the 5-HT3A-ICD is not only required but also sufficient for interaction with RIC-3. Our results suggest the ICD constitutes an oligomerization domain. This novel role significantly adds to its known contributions in receptor trafficking, targeting, and functional fine-tuning. The innate diversity of the ICDs with sizes ranging from 50 to 280 amino acids indicates new methodologies need to be developed to determine the structures of these domains. The use of soluble ICD proteins that we report in the present study constitutes a useful approach to address this gap.

Delineating the site of interaction on the intracellular domain of 5-HT3A receptors with the chaperone protein RIC-3

“…The crystal structure of the 5-HT 3A contains a partially resolved ICD with three distinct segments: the MX-helix following a Figure 3A). The direct interaction of the full-length ICD and RIC-3 had been shown previously in a similar experiment and the ICD construct here was used as a control [25]. MBP alone or omitting the RIC-3 in the pull-down did not reveal the presence of the prey protein.…”

“…Resistance to inhibitors of cholinesterase (RIC-3) is a chaperon protein known to modulate maturation and membrane trafficking of some members of pLGICs. Previously we have shown that the ICD mediates this interaction [21][22][23] ,and that the ICD alone, in the absence of both ECD and TMD, interacts with RIC-3 [25].…”

“…In summary, from previous studies, it is known that the 5-HT 3A -ICD is required and sufficient for interaction of the 5-HT 3A R and RIC-3. Furthermore, this direct interaction between the ICD and RIC-3 does not require the presence of other host cell proteins [21][22][23][24][25]. In the current study, we aimed to identify the segment of the ICD responsible for this interaction with the hypothesis that the interaction site lies within an independent segment of the ICD.…”

“…Addition of MBP to the 5-HT 3A -ICD, facilitated the expression, purification and stability of this domain. We determined, using a similar pull down assay, that the purified 5-HT 3A -ICD maintains an interaction with RIC-3 [25].…”

“…Our results identified a stretch of 24 amino acids within the 115 amino acid long 5-HT 3A -ICD as the interaction site of RIC-3. fused to the C-terminus of maltose binding protein (MBP) in pMALX vector (New England Biolabs), MBP-5-HT 3A -ICD-pMALX, was used as the template to design constructs for the present study [25,29]. The fusion constructs were generated by deletion of amino acids from [24,31,32].…”