Figure 4.

Differential effects of Rab43N131I on the surface expression of α₂-AR and mAChR in neuronal SHSY5Y and non-neuronal NRK49F cells.A, effects of Rab43N131I on the surface expression of endogenous α₂-AR and mAChR. The cells were cultured on 6-well plates and infected with control and Rab43N131I viruses for 48 h. The surface expression of α₂-AR and mAChR was determined by intact cell ligand binding as described in the legend to Figure 1B. In a typical experiment, the mean values of specific ligand binding of α₂-AR and mAChR were 2336 and 8036 DPM, respectively, in SHSY5Y cells infected with control viruses, and were 2588 and 1831 DPM, respectively, in control NRK49F cells. B, BFA treatment attenuates the surface expression of α₂-AR and mAChR in SHSY5Y and NRK49F cells. The cells were treated with DMSO or BFA at 1 μg/ml for 24 h, and the surface receptor expression was measured by radioligand binding. C, effects of Rab43 mutants on the surface expression of α_2B-AR and M3R in SHSY5Y cells as measured by BRET assays. The cells were cultured on 12-well dishes and transfected with 250 ng of Rluc–α_2B-AR or Rluc–M3R and 750 ng of venus-kRas together with 1 μg of DsRed–Rab43 or DsRed vectors. D, effects of Rab43N131I on the subcellular distribution of α_2B-AR and M3R. The cells were transfected with GFP–α_2B-AR or GFP–M3R together with DsRed vectors or DsRed–Rab43N131I. The subcellular localization of the receptors was revealed by confocal microscopy. Inserts show the expression of DsRed or DsRed–Rab43. Bars represent the mean ± SD (n = 3). ∗p < 0.05 relative to respective control. The scale bar represents 10 μm. α₂-AR, α₂-adrenergic receptor; BRET, bioluminescence resonance energy transfer; M3R, M3 mAChR; mAChR, muscarinic acetylcholine receptor.