Roles of conserved intracellular sequences regions for the proper expression of dopamine D2 and D3 receptors

The dopamine D 2 receptor and D 3 receptor (D 2 R, D 3 R) have high homology in both their amino acid composition and signaling pathways. Virtually all signaling pathways reported thus far overlap between the two receptors with the exception that the D 3 R signals are 2∼5 times less efficient than D 2 R. Previous studies have suggested that conformational constraints of D 3 R might be responsible for the poor coupling with the G protein. To this hypothesis, point mutations were introduced into some of the conserved regions between D 2 R and D 3 R, and their effects on receptor expression were investigated. Among the four conserved intracellular receptor regions examined (TTT motif in the 1 st intracellular loop, SS motif in the 2 nd intracellular loop, YxxL and TxxS/xS motifs in the 3 rd intracellular loop), a mutation of the Thr-Thr-Thr (TTT) motif in the first intracellular loop or the LxxY motif in the 3 rd intracellular loop markedly decreased the level of D 3 R expression compared with D 2 R. The TTT motif was further mutated individually or in combination to test which residue plays a critical role on the expression of the receptor proteins. Different amino acids between D 2 R and D 3 R in the 1 st intracellular loop were exchanged to determine if the adjacent amino acid residues are responsible for the differences between D 2 R and D 3 R. The first two threonine residues become more important when the individual threonine residue is mutated. However, all three intact threonine residues are essential for proper expression of the receptor proteins. The neighboring sequences around the triplet threonine residues in the 1 st loop of D 3 R are not important for proper positioning of the receptor proteins on the plasma membrane. It was concluded that D 2 R has a more flexible overall conformation that can accept mutated residues in the intracellular region than D 3 R, which might be partly responsible for the quantitative differences in the signaling efficiency between D 2 R and D 3 R.