Genome‐wide tissue transcriptome profiling and ligand screening identify seven striatum‐specific human orphan GPCRs
Authors: Allen John A , Raval Sweta R , Yang Robert , Xi Simon 
Published: November 2023

The striatum serves as the primary input of the basal ganglia performing essential brain functions including movement control, regulation of attention and motivation and is a critical component of the reward system. Of the approximately 350 non‐sensory human G protein‐coupled receptors (GPCRs), roughly 120 remain orphan receptors whose endogenous ligands and physiological functions are largely unknown. Here we employed a genome‐wide tissue transcriptomic analysis to identify GPCRs selectively expressed in the human striatum and utilized a high throughput screening platform to search for ligands active at striatal orphan receptors. We conducted a comprehensive survey of human tissue‐selective gene expression using an unprecedented collection of 1640 high‐quality RNA‐seq samples from the Genotype Tissue Expression (GTEx) pilot project, covering 31 human peripheral tissues and 13 brain subregions, including the caudate, putamen and nucleus accumbens. We developed a weighted tissue‐selectivity scoring method that takes into account the similarity and difference of gene expression in all tissues and variability across individual samples. To identify receptor activity and screen for new pharmacology, human clones of receptors were expressed in HEK293 cells and signaling assessed using the PRESTO‐Tango beta arrestin assay or the Glo‐sensor cAMP assay. As a screening proof‐of‐concept validation, the PRESTO‐Tango assay was used to screen the LOPAC library of 1280 pharmacologically active small molecules against the striatal orphan receptors. The gene expression analysis identified a total of 99 protein‐coding genes and 76 non‐coding RNAs selectively expressed in the human striatum. Functional annotation of striatal selective protein‐coding genes showed strong enrichment in Dopamine neurotransmission and signaling (e.g. DRD1, DRD2, DRD3, PDE1B, PDE10A, PPP1R1B, RGS14), consistent with the well‐described function of the striatum to coordinate dopamine‐dependent brain functions. Strikingly, a large number of GPCRs were selectively expressed in the human striatum with 18 out of the 99 striatal selective genes identified as encoding GPCRs. Out of these GPCRs, 11 have known ligands including many established therapeutic or investigational drug targets (e.g. D1R, D2R) and seven of the receptors identified were class A orphans, namely, GPR6, GPR52, GPR55, GPR88, GPR101, GPR139 and GPR149. When expressed in HEK293 cells, the human striatal orphan receptors exhibited varying degrees of basal/constitutive activity for altering cAMP levels with GPR6, GPR52 and GPR101 increasing cAMP suggesting Gs/olf coupling, while GPR88 decreased cAMP levels suggesting Gi/o coupling. Preliminary screening results from the PRESTO‐Tango platform indicated all orphan receptors were appropriately plasma membrane localized and they induced varying degrees of basal/constitutive activity to activate beta‐arrestins. Although future pharmacological and physiological studies are required and ongoing, identification of this large number of orphan receptors selectively expressed in the human striatum suggests these receptors may modulate striatal neurotransmission and they function to regulate the basal ganglia. Our findings also suggest the seven identified human orphan GPCRs are potential drug targets amenable to screening whose pharmacological modulation may be therapeutic for treating striatum‐related neurological diseases.Support or Funding InformationCenter for Addiction Research at UTMB and The University of Texas System