Structural and functional analysis of RIC8B variants regulating olfactory receptor responses

Resistance to inhibitors of cholinesterase 8B (RIC8B) functions as a chaperone and guanine nucleotide exchange factor for Gαs/olf. We focused on RIC8B variant 1 (v1), which is abundantly expressed at the mRNA level, and variant 4 (v4), which lacks the C-terminal cradle loop helix (CLH) domain. Together with 3 closely related variants (v2, v3, and v10), we evaluated 5 variants for chaperone activity. HEK293T cells were co-expressed with olfactory receptors (ORs), and odorant-induced cAMP production was used as a functional readout. Among the variants tested, only v4 consistently suppressed cAMP responses. AlphaFold3-based complex structure prediction indicated that v1 forms multiple hydrogen bonds with Gαs via its CLH domain, whereas v4 failed to establish these interactions. This suggests that v4 may be unable to stably adopt the proper binding conformation with Gαs, potentially resulting in improperly folded Gαs that exert a dominant-negative effect on OR responses.