Figure 6.

Rab43N131I and Rab43KO inhibit the cell surface transport of M3-2B from the ER in cells.A, differential interaction of the ICL3 of α_2B-AR and M3R with Rab43 in GST fusion protein pull-down assays. Similar results were obtained in three individual experiments. B, a diagram showing the generation of the chimeric receptor M3-2B in which the M3R ICL3 was substituted with the α_2B-AR ICL3 which binds to Rab43. C, Rab43 interaction with α_2B-AR, M3R, and M3-2B in live cells in BRET assays. D, inhibition of the surface expression of M3-2B by Rab43N131I and Rab43KO as measured by radioligand binding assays using [³H]-NMS at 20 nM. E, effects of Rab43N131I on the subcellular distribution of M3-2B. The cells were transfected with M3-2B–GFP together with DsRed vectors or DsRed–Rab43N131I. F, effects of Rab43KO by CRISPR–Cas9 on the subcellular distribution of M3-2B. The cells were transfected with M3-2B–CFP and GFP-carrying CRISPR-Cas9 control vectors or KO plasmids targeting human Rab43 (SHSY5Y cells) or mouse Rab43 (NRK49F cells). G and H, colocalization of M3-2B with calregulin in cells expressing Rab43N131I (G) or Rab43KO (H). The cells were transfected with M3-2B–CFP and GFP–Rab43N131I (G) or GFP-carrying Rab43KO plasmids (H) and then stained with calregulin antibodies. Inserts show DsRed (E) or GFP (F). Bars represent the mean ± SD (n = 3). The scale bars represent 10 μm. [³H]-NMS, [N-methyl-³H]-scopolamine methyl chloride; α₂-AR, α₂-adrenergic receptor; BRET, bioluminescence resonance energy transfer; CFP, cyan fluorescent protein; ER, endoplasmic reticulum; ICL3, third intracellular loop; M3-2B, M3R mutant containing the α_2B-AR ICL3; M3R, M3 mAChR.