Biology subjects

Kapolka, N. J.

Publications and source records attributed to Kapolka, N. J..

3 recordsLinked to original sources

bioRxiv2026

Systematic mapping of chimera-permissive sites by CRISPR-guided PAM scanning

Engineered protein chimeras enable new biological functions but remain difficult to design due to context-dependent constraints on insertion tolerance and the need to preserve host protein function. Here, we report CRISPR-guided protospacer adjacent motif (PAM) scanning in yeast to map chimera-permissive sites in living cells. We apply this approach to peptide and reporter insertions. In the first application, we generated 91 insertion chimeras encoding a defined protease cleavage sequence across six components of a model G protein-coupled receptor (GPCR) signaling pathway. Sixty-three percent of sites retain signaling, identifying positions that preserve host function and reveal broad, position-dependent tolerance. Coupling insertional scanning with cognate proteases enables site-resolved mapping of in-cell accessibility, distinguishing protected and exposed regions and defining EX1- and EX2-like regimes. These chimeras are responsive to proteolysis and pharmacological inhibition, enabling reversible control of protein activity. In a second application, we scanned 32 positions in yeast Ste2 and human A2A and MTNR1A receptors to engineer bi-functional chimeras that retain native function while incorporating reporter activity. Together, these results establish PAM scanning as a scalable, protein-agnostic framework for mapping insertion tolerance, interrogating protein accessibility in vivo, and enabling scalable ground-truth benchmarking of predictive chimera engineering.

synthetic biology↗

bioRxiv2021

Proton-gated coincidence detection is a common feature of GPCR signaling

The evolutionary expansion of G protein-coupled receptors (GPCRs) has produced a rich diversity of transmembrane sensors for many physical and chemical signals. In humans alone, over 800 GPCRs detect stimuli such as light, hormones, and metabolites to guide cellular decision making primarily using intracellular G protein signaling networks. This diversity is further enriched by GPCRs that function as molecular logic gates capable of discerning multiple inputs to transduce cues encoded in complex, context-dependent signals. Here, we show that many GPCRs are switch-like Boolean-gated coincidence detectors that link proton (H+) binding to GPCR signaling. Using a panel of 28 receptors, covering 280 individual GPCR-G coupling combinations, we show that H+ gating both positively and negatively modulates and controls GPCR signaling. Notably, these observations extend to all modes of GPCR pharmacology including ligand efficacy, potency, and cooperativity. Additionally, we show that GPCR antagonism and constitutive activity are regulated by H+ gating and report the discovery of a new acid sensor, the adenosine A2a receptor (ADORA2A), which can be activated solely by acidic pH. Together, these findings establish a new paradigm for GPCR biology and pharmacology in acidified microenvironments such as endosomes, synapses, tumors, and ischemic vasculature.

pharmacology and toxicology↗

bioRxiv2020

DCyFIR: a high-throughput CRISPR platform for multiplexed G protein-coupled receptor profiling and ligand discovery

More than 800 G protein-coupled receptors (GPCRs) comprise the largest class of membrane receptors in humans. While there is ample biological understanding and many approved drugs for prototypic GPCRs, most GPCRs still lack well-defined biological ligands and drugs. Here, we report our efforts to tap the potential of understudied GPCRs by developing yeast-based technologies for high-throughput CRISPR engineering and GPCR ligand discovery. We refer to these technologies collectively as Dynamic Cyan induction by Functional Integrated Receptors: DCyFIR. A major advantage of DCyFIR is that GPCRs and other assay components are CRISPR-integrated directly into the yeast genome, making it possible to decode ligand specificity by profiling mixtures of GPCR-barcoded yeast strains in a single tube. To demonstrate the capabilities of DCyFIR, we engineered a yeast strain library of 30 human GPCRs and their 300 possible GPCR-G coupling combinations. Profiling of these 300 strains, using parallel (DCyFIRscreen) and multiplex (DCyFIRplex) DCyFIR modes, recapitulated known GPCR agonism with 100% accuracy, and identified unexpected interactions for the receptors ADRA2B, HCAR3, MTNR1A, S1PR1, and S1PR2. To demonstrate DCyFIR scalability, we profiled a library of 320 human metabolites and observed new GPCR-ligand interactions with amino acid, lipid, sugar, and steroid metabolites. Remarkably, many of these findings pertained to understudied "pharmacologically dark" receptors GPR4, GPR65, GPR68, and HCAR3. For example, we found that kynurenic acid activated HCAR3 with a nearly 20-fold lower EC50 than GPR35, its known receptor. Taken together, these findings demonstrate the power of DCyFIR for identifying novel ligand interactions with prototypic and understudied GPCRs. Significance StatementG protein-coupled receptors (GPCRs) are the largest class of membrane receptors in humans. As such, GPCR signaling is central to human biology and medicine. While more than 30% of approved drugs target roughly 150 GPCRs, most receptors lack well-defined endogenous ligands and are currently not druggable. To address this challenge, we created a GPCR screening platform for ligand and drug discovery. This innovative technology enables the cost-effective profiling of ligands and drug compounds against mixtures of hundreds of GPCR-barcoded cell strains in a single experiment. Because a ligand or drug is tested against a collection of receptors all at once, our novel method accelerates the process of identifying potential GPCR ligands and drugs.

pharmacology and toxicology↗