Structural and functional analysis of RIC8B variants regulating olfactory receptor responses

Area of Science:

• Molecular Biology
• Cell Signaling
• Structural Biology

Background:

• Resistance to inhibitors of cholinesterase 8B (RIC8B) is a chaperone and guanine nucleotide exchange factor for Gαs/olf.
• RIC8B has multiple variants, with variant 1 (v1) highly expressed and variant 4 (v4) lacking the C-terminal Cradle Loop Helix (CLH) domain.

Purpose of the Study:

• To evaluate the functional impact of different RIC8B variants, particularly v1 and v4, on olfactory receptor (OR) signaling.
• To investigate the structural basis for differential interactions between RIC8B variants and Gαs/olf.

Main Methods:

• Co-expression of HEK293T cells with olfactory receptors (ORs).
• Measurement of odorant-induced cAMP production as a functional readout.
• AlphaFold3-based complex structure prediction to analyze protein-protein interactions.

Main Results:

• RIC8B variant 4 (v4) consistently suppressed cAMP responses mediated by ORs, unlike other variants tested.
• AlphaFold3 predicted that v1 forms stabilizing hydrogen bonds with Gαs via its CLH domain.
• Variant v4 failed to form these critical interactions with Gαs, suggesting conformational instability.

Conclusions:

• The C-terminal CLH domain of RIC8B is crucial for stable Gαs binding and proper chaperone function.
• RIC8B variant v4's inability to properly bind Gαs may lead to misfolded Gαs, causing a dominant-negative effect on OR signaling.
• These findings highlight the importance of specific protein domains in regulating G protein signaling pathways.