Browsing by Author "Tsvetanova, Nikoleta"
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Item Open Access Discovering novel G-protein coupled receptor (GPCR) / cyclic AMP (cAMP) pathway regulators and their physiological relevance(2023) Semesta, Khairunnisa MentariG-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.
Item Embargo Getting Up Close and Personal: What Makes an Endosome Special in GPCR Signaling(2023) Willette, Blair Kristene AndersonG protein-coupled receptors (GPCRs), a class of critical regulators of mammalian physiology, can initiate unique functional responses depending on the subcellular compartment of their activation. Yet, how endosomal receptors transduce location-biased outcomes remains poorly understood. Efforts to uncover the mechanistic basis of compartmentalized GPCR signaling have largely focused on the biochemical aspect of this regulation through dissection of the relevant factors. Here, we assessed the biophysical positioning of receptor-containing endosomes as an alternative salient mechanism coordinating the transduction of spatially-biased responses. We focused on the beta2-adrenergic receptor (β2AR), a prototypical GPCR that signals from early endosomes via cyclic AMP (cAMP) production. We examined the role of endosome positioning in the context of gene transcription as a representative signaling readout, because endosomal β2ARs are well-known to selectively stimulate transcriptional reprogramming. First, we developed subcellular-localized cAMP and protein kinase A (PKA) sensors to enable selective quantification of endosomal GPCR-mediated activity. We also generated two complementary optical readouts that enable robust measurements of bulk- and gene-specific GPCR/cAMP-dependent transcription with single-cell resolution. We next overcame a technical challenge that has hindered the direct assessment of the functional role of endosome positioning by devising a strategy to selectively and rapidly redistribute receptor-containing endosomes ‘on command’ in intact cells without perturbing their biochemical composition. By combining these readouts with rapid endosome relocalization, we established that disruption of native endosome positioning inhibits the initiation of the endosome-dependent responses. Lastly, utilizing the cAMP and PKA sensors, we demonstrated a prominent mechanistic role of local PKA activity and phosphodiesterase (PDE)-mediated cAMP hydrolysis in this process. This dissertation work, therefore, illuminates a novel mechanism regulating GPCR function by identifying endosome positioning as the principal mediator of spatially-selective receptor signaling.