GPR37 and GPR37L1 are glia-enriched GPCRs that have been implicated in several neurological and neurodegenerative diseases. To gain insight into the potential molecular mechanisms by which GPR37 and GPR37L1 regulate cellular physiology, proteomic analyses of whole mouse brain tissue from wild-type (WT) versus GPR37/GPR37L1 double knockout (DKO) mice were performed in order to identify proteins regulated by the absence versus presence of these receptors (data are available via ProteomeXchange with identifier PXD015202). These analyses revealed a number of proteins that were significantly increased or decreased by the absence of GPR37 and GPR37L1. One of the most decreased proteins in the DKO versus WT brain tissue was S100A5, a calcium-binding protein, and the reduction of S100A5 expression in KO brain tissue was validated via Western blot. Co-expression of S100A5 with either GPR37 or GPR37L1 in HEK293T cells did not result in any change in S100A5 expression but did robustly increase secretion of S100A5. To dissect the mechanism by which S100A5 secretion was enhanced, cells co-expressing S100A5 with the receptors were treated with different pharmacological reagents. These studies revealed that calcium is essential for the secretion of S100A5 downstream of GPR37 and GPR37L1 signaling, as treatment with BAPTA-AM, an intracellular Ca 2+ chelator, reduced S100A5 secretion from transfected HEK293T cells. Collectively these findings provide a panoramic view of proteomic changes resulting from loss of GPR37 and GPR37L1 and also impart mechanistic insight into the regulation of S100A5 by these receptors, thereby shedding light on the functions of GPR37 and GPR37L1 in brain tissue.

The D1 dopamine receptor (D1R) is a G protein-coupled receptor that signals through activating adenylyl cyclase and raising intracellular cAMP levels. When activated, the D1R also recruits the scaffolding protein β-arrestin, which promotes receptor desensitization and internalization, as well as additional downstream signaling pathways. These processes are triggered through receptor phosphorylation by G protein-coupled receptor kinases (GRKs), although the precise phosphorylation sites and their role in recruiting β-arrestin to the D1R remains incompletely described. In this study, we have used detailed mutational and in situ phosphorylation analyses to completely identify the GRK-mediated phosphorylation sites on the D1R. Our results indicate that GRKs can phosphorylate 14 serine and threonine residues within the C-terminus and the third intracellular loop (ICL3) of the receptor, and that this occurs in a hierarchical fashion, where phosphorylation of the C-terminus precedes that of the ICL3. Using β-arrestin recruitment assays, we identified a cluster of phosphorylation sites in the proximal region of the C-terminus that drive β-arrestin binding to the D1R. We further provide evidence that phosphorylation sites in the ICL3 are responsible for β-arrestin activation, leading to receptor internalization. Our results suggest that distinct D1R GRK phosphorylation sites are involved in β-arrestin binding and activation.

Poster Board 281Dopamine receptors (DARs) are G-protein coupled receptors (GPCRs) that regulate diverse physiological functions including cognition, mood, movement, and reward-related behaviors, and are involved in the treatment or etiology of many neuropsychiatric disorders including schizophrenia and substance use disorder (SUD). DARs are classified as either D1-like (D1R and D5R) or D2like (D2R, D3R, and D4R) based on structural homology and pharmacological profiles. Antagonists of D2-like DARs are currently used in the therapies for many neuropsychiatric disorders, but D3R-selective antagonists may be better therapeutics for schizophrenia or SUD as they could attenuate psychotic or drug craving symptoms without the motor side effects frequently produced by D2R-preferring antagonists due to limited distribution of the D3R in the brain. However, discovery of D3R-selective compounds is challenging due to high sequence homology of the D2R and D3R within their orthosteric binding sites, leading to the potential for off-target side effects produced by currently available compounds due to simultaneous antagonism of both subtypes or other closely related receptors. Our lab has endeavored to overcome the selectivity challenges posed by orthosteric antagonists by utilizing a D3R-mediated b-arrestin recruitment assay to screen the NIH Molecular Libraries Program 400,000+ small molecule library for compounds that inhibit the D3R via binding to less conserved allosteric sites. The most potent hit compound, MLS6357, was selective for the D3R versus the D2R and D4R in several functional outputs including b-arrestin recruitment and G-protein activation. Radioligand binding and functional assays using closely related GPCRs revealed that MLS6357 has very limited cross-reactivity with other GPCRs. Additionally, Schild-type functional assays showed that MLS6357 acts as a purely non-competitive negative allosteric modulator (NAM) of the D3R. We synthesized and characterized > 70 analogs of MLS6357 using iterative medicinal chemistry approaches which produced analogs that are 100-fold and 60-fold more potent than the parent compound in D3R-mediated b-arrestin recruitment and G-protein activation assays, respectively, and revealed structure-activity relationships for further optimization of the scaffold. Moreover, some analogs appear to display functional selectivity for inhibition of G-protein activation versus inhibition of b-arrestin recruitment, and vice versa, and some also display inverse agonist activity in G-protein signaling assays. Using in vivo pharmacokinetic experiments in mice via i.p. administration, one of the lead analogs was found to be brain penetrant and achieved sufficient concentrations to occupy the D3R in vivo. To identify the allosteric binding site for the MLS6357 scaffold on the D3R, we utilized various D3R/D2R chimeras, receptor mutants, and molecular modeling techniques to reveal and characterize receptor regions necessary for compound efficacy. Further refinement of the binding pocket for MLS6357 will ...

Dopamine receptors (DARs) are G protein‐coupled receptors (GPCRs) that regulate diverse physiological functions and are involved in the treatment and/or etiology of many neuropsychiatric disorders including schizophrenia and substance use disorder (SUD). DARs are classified as either D1‐like (D1R and D5R) or D2‐like (D2R, D3R, and D4R) based on structural homology and pharmacological profiles. Antagonists of D2‐like DARs are currently used in the therapies for many neuropsychiatric disorders. D3R‐selective antagonists have the potential to be better therapeutics for schizophrenia or SUD as they could attenuate psychotic or drug craving symptoms without the motor side effects frequently produced by D2R‐preferring antagonists. Unfortunately, discovery of subtype‐selective compounds for the D3R and D2R has been challenging due to high sequence homology within the orthosteric binding sites of DARs. However, compounds that modulate receptor activities through interactions with less conserved allosteric sites have the potential to be highly selective. To find highly selective allosteric antagonists of the D3R, we screened the NIH Molecular Libraries Program 400,000+ small molecule library with a D3R‐mediated β‐arrestin recruitment assay. We found that one compound, MLS6357, was selective for the D3R over the D2R and D4R in several functional outputs including β‐arrestin recruitment and G‐protein activation. Radioligand binding and functional assays using closely related GPCRs revealed that MLS6357 has very limited cross‐reactivity with other GPCRs. Additionally, Schild‐type functional assays showed that MLS6357 acts as a purely non‐competitive negative allosteric modulator (NAM) of the D3R. We synthesized and characterized >60 analogs of MLS6357 using iterative medicinal chemistry approaches, which revealed structure–activity relationships and enabled further optimization of the scaffold. These efforts produced analogs that are 10‐fold and 30‐fold more potent than the parent compound in D3R‐mediated β‐arrestin recruitment and G‐protein activation assays, respectively. Moreover, some analogs appear to display functional selectivity for inhibition of G‐protein activation versus inhibition of β‐arrestin recruitment, and vice versa, and some also display inverse agonist activity. To identify the allosteric binding site for the MLS6357 scaffold on the D3R, we utilized various D3R/D2R chimeras, receptor mutants, and molecular modeling techniques to reveal and characterize receptor regions necessary for compound efficacy. Further refinement of the binding pocket for MLS6357 will inform future medicinal chemistry efforts. Ultimately, this novel scaffold may be of benefit as a pharmacological probe or therapeutic lead for D3R‐related pathophysiology.