Discovering novel G-protein coupled receptor (GPCR) / cyclic AMP (cAMP) pathway regulators and their physiological relevance

Abstract

G-protein coupled receptors (GPCRs) are pivotal to multiple physiological processes, including neuronal functions such as neurotransmission and memory formation. Consequently, aberrant GPCR signaling has been implicated in complex psychiatric disorders. Our ability to develop efficient therapeutic strategies depends on our understanding of the molecular factors that govern GPCR activity. We performed a genome-wide CRISPR interference screen using a fluorescent cAMP-dependent transcriptional reporter and identified 95 novel regulators that had no previous connection to the GPCR/cAMP pathway. We functionally validated eight regulators and showed that they control distinct steps of the pathway. One of these, RNA binding motif 12 (RBM12), is a novel potent negative regulator of the GPCR/cAMP pathway. Truncating RBM12 mutations are implicated in highly penetrant schizophrenia and defective brain development, yet its cellular function is unknown. To investigate its role in GPCR/cAMP signaling, we performed CRISPR-based gene editing in two complementary cellular models: experimentally tractable HEK293 cells and physiologically relevant human induced pluripotent stem cell-derived neurons. We found that RBM12 deficiency leads to hyperactive cAMP accumulation, PKA activity, and downstream CREB-dependent transcriptional responses in response to the activation of stimulatory GPCRs with key functions in neurobiology (beta-adrenergic, dopamine, and adenosine). We observed that the cAMP signaling and PKA activity are independently subject to regulation by RBM12. This signaling hyperactivity is conserved in the two cellular model systems. We further show that schizophrenia-associated truncating RBM12 mutations (c.2377G>T and c.2532delT) failed to rescue the signaling hyperactivation due to possible loss-of-function and protein stability defect. In agreement with its activity as an RNA-binding protein, we show that RBM12 depletion led to altered expression of genes with established roles in the signaling pathway, including an upregulation of cyclases and the protein kinase A catalytic subunits as well as a downregulation of phosphodiesterases. In addition, we observed aberrant gene expression in neurogenesis, neurodevelopment, and differentiation pathways. Together, these experiments provide critical insights into this uncharacterized gene and its function as a novel regulator of GPCR signaling. By uncovering the novel regulatory role of a schizophrenia-risk gene in GPCR signaling, we expand our understanding of the molecular basis of neuropsychiatric disorders and enable the identification of novel druggable targets.