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. 2016 Apr 12;15(12):3665–3684. doi: 10.1074/mcp.M114.047514

Fig. 5.

Fig. 5.

Novel subunit-distinctive PTM signatures discovered herein likely underpin high subunit-biases in hGABAAR assembly and ligand interactions. A, Novel PTM signatures for GABAAR subunits include: high-occupancy N-glycosylations in nontop-surface mid-ECD, and tentative diverse partial-occupancy K-Me/Ac in ICL2, in nonstimulated cells. TM Cys also displayed subunit-specific distribution patterns (supplemental Fig. S4C). 3D locations of mid-ECD N-glycosylations in GABAAR subunits were proposed by sequence alignment with 3RHW and 2QC1. N-glycans were superimposed to mid-ECD sites identified with high occupancy. B, Top and side views of proposed physical form of functional (α1)2(β3)2(γ2L)1 GABAAR pentamer completed with mid-ECD N-glycosylations. Top-surface N-glycans were not shown. C, Glycans shown for mid-ECD sites in hGABAAR/HEK293 were deduced by trimming that resolved in 2QC1 to (GlcNAc)2(Man)5. Immediate ligand-interacting saccharides resolved in 2QC1 were shown in red. D, Subunit interfaces in classic representation of clockwise αβαβγ GABAAR (left) can be upgraded with mid-ECD N-glycans (right, red border) to illustrate concepts of heterogeneity. E, Screening Cys-loop LGICs for NX(!P)S/T motif pinpointed 8 human mid-ECD coding regions from ECD sequence, based on alignment to 3RHW (diagram). Four mains involving αβγ-hGABAAR (red) are: A1, β4-β5 loop on inner surface (α); B, C-C loop (β); C1, preloop C mid-β9 (γ); C2, upper-β9 (β3 s site NVV). Four minors: D, β8-β8 loop; E, β8-β9 lower-loop F; F, upper-β10; G, lower-β10. TMD-originated sites for ECD-TMD interface regions: TM2/TM3 linker (T1) and TM4 tail (T2) (supplemental Fig. S4B). Red box, high-occupancy sites identified herein or by 3RHW and 2QC1; pink box, NVV. Gray, A2 is likely back to top surface (β5-β6, supplemental Fig. S4A) and T0 (TM1 front) is yet unseen though a potential site (** 0, because one NPS in ELIC). * One NAS in C. elegans GluCl β subunit but not in the 3RHW crystallized α-based construct. # One NGT on tip of loop C in fish Tm nAChR chain C (2BG9), absent in human.