The tolerance property of human D3 dopamine receptor is determined by specific amino acid residues in the second cytoplasmic loop.

The D2 and D3 dopamine receptor subtypes are structurally homologous and couple to the same signal transduction pathways. Nevertheless, their evolutionary conservation suggests that the two subtypes might exhibit unique signaling characteristics. We previously determined that D3 but not D2S dopamine receptor exhibits a tolerance property in which the D3 receptor-activated G-protein coupled inward rectifier potassium currents progressively decreases upon repeated agonist stimulation. In this paper, using AtT-20 neuroendocrine cells stably expressing either human D3 or D2S receptor, we show that the tolerance property is also observed in the D3 receptor-adenylyl cyclase and D3 receptor-mitogen-activated protein kinase signaling pathways. We have previously shown that the second cytoplasmic loop of D3 receptor is required for tolerance. Here, using site-directed mutagenesis, we identified the specific amino acids in the D3 second cytoplasmic loop involved in the tolerance property. The results show that substitution of a non-conserved cysteine residue at position 147 with positively-charged lysine or arginine residues abolishes tolerance. Interestingly, the cysteine 147 residue is embedded in a putative phosphorylation site adjacent to two serine residues. Mutation of these serine residues to alanine also attenuates tolerance. Taken together, these structural studies suggest a role for phosphorylation in D3 receptor tolerance property.