FIGURE 4.

Transmembrane and cytoplasmic domains from both DRα and DRβ are independently targeted by MARCH I and MARCH VIII. CD8 reporter constructs comprising the CD8 extracellular domain and DRα or DRβ transmembrane and cytoplasmic tails were stably expressed in Mel JuSo cells (A) or transiently expressed in 293T cells (B). Dot plots show GFP (FL1) on the x axis and anti-CD8 (PE-OKT8) binding (FL2) on the y axis. The R3 gate, which represents cells expressing the E3 ligase, as assessed by GFP expression, was set against IgG control at 0.1%. The IgG control was set using cells expressing GFP (R3), to enable direct comparison between E3 ligase-expressing transfectants. Data are representative of at least three independent experiments. A, a CD8 chimera containing the transmembrane and cytoplasmic tail of DRβ is subject to MARCH I- and MARCH VIII-induced down-regulation. This was abolished by substitution of lysine residue Lys²²⁵ for arginine (CD8-DRB-K225R). The dileucine motif at positions 235 and 236 was not required for MARCH-induced down-regulation, since the behavior of CD8-DRB-L235A,L236A was indistinguishable from that of CD8-DRB. B, CD8-DRA was also targeted by MARCH I and MARCH VIII, and this was dependent upon a lysine residue, Lys²¹⁹, in its cytoplasmic tail. The extent of surface down-regulation of CD8-DRA was less than for CD8-DRB. Data are representative of at least three independent experiments.