Fig. 6.
Surface expression of myc- and HA-tagged GABAB receptor mutants. A, Surface expression in transfected HEK293. The C-terminal deletion mutant R1L921 is expressed at the cell surface, whereas R1P928 is retained intracellularly. Substitution of alanine for R922(R1[ASRRHPP]), R924(R1[RSARHPP]), and to a lesser extent R925 (R1[RSRAHPP]) results in surface expression of GABAB(1), whereas substitution of S923 (R1[RARRHPP]) does not affect intracellular retention. A C-terminal fragment of GABAB(2) encoding residues R714 through P820 (CR2P820) efficiently traffics WT GABAB(1) to the plasma membrane. R2L791 with a more extensive C-terminal deletion is unable to deliver WT GABAB(1) to the surface. Removal of the LZ1 is sufficient to direct R1ΔLZ1 to the cell surface, indicating that LZ1 is required for correct exposure of the ERR/R signal. R1LZ2 does not reveal surface immunoreactivity but is detectable at the plasma membrane on coexpression with either R2LZ1 or WT GABAB(2). R1[PPP] does not show significant surface immunoreactivity when expressed alone but is targeted to the plasma membrane on coexpression with either WT GABAB(2) or R2[PPP]. Coexpression with R2T749 is insufficient to traffic R1[PPP] to the cell surface, because no shielding of the fully functional ERR/R in R1[PPP] is provided. For detection of intracellular myc and HA epitopes, (permeabilized) transfected cells were permeabilized with saponin. B, Surface expression in transfected primary cerebellar granule cells. Neurons transfected with myc-tagged WT GABAB(1) (R1) alone do not show cell surface immunoreactivity. Strong c-myc surface staining is detected with cells cotransfected with WT GABAB(1) and GABAB(2)(R1+R2) and with mutant R1[ASARHPP]. For surface labeling, transfected cerebellar neurons were incubated with anti-c-myc antibody in culture medium for 1 hr, washed, and fixed with paraformaldehyde. Neurons were incubated for 1.5 hr with anti-mouse IgG Texas Red conjugate (1:1000), washed extensively, mounted on coverslips, and viewed with a 63× magnification objective. For labeling of permeabilized cells, paraformaldehyde-fixed neurons were permeabilized with 0.05% Triton X-100 before incubation with primary and secondary antibodies. Three independent experiments were performed.