An amino acid residue in the second extracellular loop determines the agonist-dependent tolerance property of the human D3 dopamine receptor.

The D3 dopamine receptor is a therapeutic target for treating various nervous system disorders such as schizophrenia, Parkinson's disease, depression, and addictive behaviors. The crystal structure of the D3 receptor bound to an antagonist was recently described; however, the structural features that contribute to agonist-induced conformational changes and signaling properties are not well understood. We have previously described the conformation-dependent tolerance and slow response termination (SRT) signaling properties of the D3 receptor and identified the C147 residue in the second intracellular loop (IL2) of the D3 receptor as important for the tolerance property. Interestingly, while IL2 and the C147 residue, in particular, were important for dopamine- and quinpirole-induced tolerance, this residue did not affect the severe tolerance induced by the high affinity, D3 receptor-selective agonist, PD128907. Here, we used D2/D3 receptor chimeras and site-specific D3 receptor mutants to identify another residue, D187, in the second extracellular loop (EC2) of the human D3 receptor that mediates the tolerance property induced by PD128907, quinpirole, pramipexole, and dopamine. Molecular dynamics simulations confirmed the distinct conformation adopted by D3 receptor during tolerance and suggested that in the tolerant D3 receptor the D187 residue in EC2 forms a salt bridge with the H354 residue in EC3. Indeed, site-directed mutation of the H354 residue resulted in loss of PD1287907-induced tolerance. The mapping of specific amino acid residues that contribute to agonist-dependent conformation changes and D3 receptor signaling properties refines the agonist-bound D3 receptor pharmacophore model which will help develop novel D3 receptor agonists.

Development of tolerance in D3 dopamine receptor signaling is accompanied by distinct changes in receptor conformation.

The D3 but not D2 dopamine receptors exhibit a tolerance property in which agonist-induced D3 receptor response progressively decreases upon repeated agonist stimulation. We have previously shown that the D3 receptor tolerance property is not mediated by receptor internalization, persistent agonist binding or a decrease in receptor binding affinity. In this paper, we test the hypothesis that alterations in D3 receptor conformation underlie the tolerance property. Structural models of wild type and mutant human D3 receptors were generated using the beta adrenergic receptor crystal structure as a template. These models suggested that the agonist-bound D3 receptor undergoes conformational changes that could underlie its tolerance property. To experimentally assess changes in receptor conformation, we measured the accessibility of native cysteine residues present in the extracellular and transmembrane regions of the human D3 receptor to two different thiol-modifying biotinylating reagents. The accessibilities of the native cysteine residues present in the D3 receptor were assessed under control conditions, in the presence of agonist and under conditions that induced receptor tolerance. By comparing the accessibility of D3 receptor cysteine residues to hydrophobic and hydrophilic thiol-modifying biotinylating reagents, we show that the alteration of D3 receptor conformation during tolerance involves the net movement of cysteine residues into a hydrophobic environment. Our results show that the conformation state of the D3 receptor during tolerance is distinct from the conformation under basal and agonist-bound conditions. The results suggest that the D3 receptor tolerance property is mediated by conformational changes that may uncouple the receptor from G-protein signaling.