Elucidating Signaling Mechanisms and Pharmacology for the Brain Orphan Receptor GPR37

Abstract ID 55957 Poster Board 487 Andrew Frazier and John A. Allen Center for Addiction Research, Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX USA Corresponding author: John A. Allen, Ph.D., Department of Pharmacology and Toxicology, 301 University Blvd., University of Texas Medical Branch, Galveston, TX, Email: joaallen@utmb.edu GPR37 is a class-A orphan G protein-coupled receptor that is most highly expressed in the human spinal cord with lesser expression in other brain regions including the amygdala, basal ganglia, hippocampus, and frontal cortex. GPR37 has been identified as a promising target for multiple conditions such as Parkinson's disease, inflammatory pain, ischemic stroke, and some forms of cancer. GPR37 is suggested to modulate cellular signaling through the activation of G_i/o signaling pathways; however, this has not been fully verified, and a natural ligand for GPR37 has yet to be identified. Here, we sought to elucidate GPR37 cellular signaling pathways by assessing basal or agonist-stimulated changes in cAMP levels using the Glosensor assay, release or influx of calcium using a real-time fluorescence imaging plate reader (FLIPR) assay, and β-arrestin2 recruitment using the Tango assay. Through these assays, we discovered that transient expression of wild-type human GPR37 in HEK293 cells significantly decreases cAMP levels in a pertussis toxin-insensitive manner, suggesting a potential signaling mechanism involving the activation of G_z but not G_i/o. When expressed in HEK293 cells, or in human oligodendroglioma (HOG) cells, wildtype human GPR37 and the mature N-terminally truncated form of GPR37, equally reduced cAMP levels. In addition, we determined that GPR37 expression strongly recruited β-arrestin2. These cellular studies also determined two previously suggested GPR37 agonists, TX14A and NPD1, fail to induce calcium signaling using the robust FLIPR assay, in opposition to previous reports. TX14A and NPD1 also failed to show a robust activation or inhibition of cAMP signaling, but both compounds decreased basal GPR37 β-arrestin2 recruitment. The information attained from this work suggests that human GPR37 is highly constitutively active to reduce cAMP signaling and to recruit β-arrestin. Further research is needed to confirm additional GPR37 signaling mechanisms and to verify if TX14A and NPD1 are activators or inhibitors of this orphan receptor. Acknowledgments: The UTMB Center for Addiction Research Support/Funding Information: NIH NINDS R61NS12728