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 agonistinduced 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. KEYWORDS: Structure−function, signal transduction, desensitization, functional selectivity, D3 receptor conformation, potassium channels T he D3 dopamine receptor is one of five dopamine receptor subtypes and belongs to the "D2-like" dopamine receptor subfamily that includes the D2 and D4 dopamine receptors. D3 receptor ligands are clinically efficacious in treating Parkinson's disease, schizophrenia, depression and addictive behaviors, making the D3 receptor an important therapeutic target.1−4 An antagonist-bound crystal structure of D3 dopamine receptor was recently published and identified domains involved in interactions with antagonists.5 Pharmacophore models based on the antagonist-bound D3 receptor structure while successful at identifying novel high affinity D3 receptor antagonists unfortunately are not ideal for identifying novel D3 receptor agonists. 6 In the absence of an agonist-bound crystal structure of the D3 receptor, the residues and domains involved in mediating agonist-dependent conformation changes in the D3 receptor are less well understood. Furthermore, with the recent discovery of functionally selective ligands that produce distinct receptor conformation which direct the coupling of the receptor to specific signaling pathways, it has become imp...

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Dopamine is a predominant neurotransmitter and improper regulation of dopaminergic transmission results in disorders such as Parkinson's disease, schizophrenia and addiction. Dopamine receptors are G‐protein coupled receptors (GPCRs) and are classified as D1‐D5. Although the D3 receptor (D3R) shares high homology with D2, it possesses distinct signaling properties when repeatedly stimulated with agonist. The D3R exhibits two unique signaling properties, tolerance and slow response termination (SRT) to Dopamine and classical D2‐like receptor agonists. Tolerance is defined as a reduction in signaling response upon a second exposure to agonist and SRT is seen as a slow termination of the response upon agonist removal. GPCRs interact with a variety of proteins including but not limited to their cognate G‐protein, regulatory and modulatory proteins such as Regulator of G protein Signaling proteins, Kinases and Phosphatases. The molecular mechanisms underlying the D3R tolerance and SRT are unknown. We hypothesized that the D3R and D2R interacts with a different set of proteins and differences in this signaling complex contributes to the phenomena of Tolerance and SRT. Our results suggest that the D2R and D3R couple to different Gαi/o proteins, suggesting a role for the G proteins in the D3R tolerance and SRT properties. Sucrose density gradient experiments indicated that the molecular mass of the D3R is different in the tolerant and non‐tolerant states while Mass Spectrometry analysis identified specific proteins interacting with the D3R in its tolerant state.