Location-biased activation of the proton-sensor GPR65 is uncoupled from receptor trafficking.

The canonical view of G protein-coupled receptor (GPCR) function is that receptor trafficking is tightly coupled to signaling. GPCRs remain on the plasma membrane (PM) at the cell surface until they are activated, after which they are desensitized and internalized into endosomal compartments. This canonical view presents an interesting context for proton-sensing GPCRs because they are more likely to be activated in acidic endosomal compartments than at the PM. Here, we show that the trafficking of the prototypical proton-sensor GPR65 is fully uncoupled from signaling, unlike that of other known mammalian GPCRs. GPR65 internalizes and localizes to early and late endosomes, from where they signal at steady state, irrespective of extracellular pH. Acidic extracellular environments stimulate receptor signaling at the PM in a dose-dependent manner, although endosomal GPR65 is still required for a full signaling response. Receptor mutants that were incapable of activating cAMP trafficked normally, internalize and localize to endosomal compartments. Our results show that GPR65 is constitutively active in endosomes, and suggest a model where changes in extracellular pH reprograms the spatial pattern of receptor signaling and biases the location of signaling to the cell surface.

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 sensors capable of discerning multiple inputs to transduce cues encoded in complex, context-dependent signals. Here, we show that many GPCRs are coincidence detectors that couple proton (H+) binding to GPCR signaling. Using a panel of 28 receptors covering 280 individual GPCR-Gα coupling combinations, we show that H+ gating both positively and negatively modulates 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 an acid sensor, the adenosine A2a receptor, which can be activated solely by acidic pH. Together, these findings establish a paradigm for GPCR signaling, biology, and pharmacology applicable to acidified microenvironments such as endosomes, synapses, tumors, and ischemic vasculature.

The evolution and mechanism of GPCR proton sensing.

Of the 800 G protein-coupled receptors (GPCRs) in humans, only three (GPR4, GPR65, and GPR68) regulate signaling in acidified microenvironments by sensing protons (H+). How these receptors have uniquely obtained this ability is unknown. Here, we show these receptors evolved the capability to sense H+ signals by acquiring buried acidic residues. Using our informatics platform pHinder, we identified a triad of buried acidic residues shared by all three receptors, a feature distinct from all other human GPCRs. Phylogenetic analysis shows the triad emerged in GPR65, the immediate ancestor of GPR4 and GPR68. To understand the evolutionary and mechanistic importance of these triad residues, we developed deep variant profiling, a yeast-based technology that utilizes high-throughput CRISPR to build and profile large libraries of GPCR variants. Using deep variant profiling and GPCR assays in HEK293 cells, we assessed the pH-sensing contributions of each triad residue in all three receptors. As predicted by our calculations, most triad mutations had profound effects consistent with direct regulation of receptor pH sensing. In addition, we found that an allosteric modulator of many class A GPCRs, Na+, synergistically regulated pH sensing by maintaining the pKa values of triad residues within the physiologically relevant pH range. As such, we show that all three receptors function as coincidence detectors of H+ and Na+. Taken together, these findings elucidate the molecular evolution and long-sought mechanism of GPR4, GPR65, and GPR68 pH sensing and provide pH-insensitive variants that should be valuable for assessing the therapeutic potential and (patho)physiological importance of GPCR pH sensing.

CRISPR-addressable yeast strains with applications in human G protein-coupled receptor profiling and synthetic biology.

Genome stability is essential for engineering cell-based devices and reporter systems. With the advent of CRISPR technology, it is now possible to build such systems by installing the necessary genetic parts directly into an organism's genome. Here, we used this approach to build a set of 10 versatile yeast-based reporter strains for studying human G protein-coupled receptors (GPCRs), the largest class of membrane receptors in humans. These reporter strains contain the necessary genetically encoded parts for studying human GPCR signaling in yeast, as well as four CRISPR-addressable expression cassettes, i.e. landing pads, installed at known safe-harbor sites in the yeast genome. We showcase the utility of these strains in two applications. First, we demonstrate that increasing GPCR expression by incrementally increasing GPCR gene copy number potentiates Gα coupling of the pharmacologically dark receptor GPR68. Second, we used two CRISPR-addressable landing pads for autocrine activation of a GPCR (the somatostatin receptor SSTR5) with its peptide agonist SRIF-14. The utility of these reporter strains can be extended far beyond these select examples to include applications such as nanobody development, mutational analysis, drug discovery, and studies of GPCR chaperoning. Additionally, we present a BY4741 yeast strain created for broad applications in the yeast and synthetic biology communities that contains only the four CRISPR-addressable landing pads. The general utility of these yeast strains provides an inexpensive, scalable, and easy means of installing and expressing genes directly from the yeast genome to build genome-barcoded sensors, reporter systems, and cell-based factories.

Submicron Aggregation of Chemically Denatured Monoclonal Antibody.

Isothermal chemical denaturation (ICD) has been widely used to evaluate the conformational stability of therapeutic proteins such as monoclonal antibodies. However, the chemical unfolding pathway and the subsequent aggregation of antibodies are not yet well-understood. In the present work, we conducted a systematic study on an ICD-induced aggregation of a pharmaceutical monoclonal antibody. Using dynamic light scattering, we monitored formation and growth of submicron aggregates in various buffers. Our experiments revealed a nucleation-controlled submicron aggregation of the antibody in the presence of chemical denaturant. After the unfolded protein reached a steady state, we reduced the denaturant concentration by dilution or dialysis to trigger further aggregation after ICD. In this way, we studied the pH effect on aggregation of the stressed protein after removal of denaturant. The ICD-dilution experiment provides a practical means for studying the propensity of unfolded proteins to form aggregates under various formulation conditions. This unique method allows us to control the degree of protein unfolding and the initiation of post-ICD aggregation.

Metastability Gap in the Phase Diagram of Monoclonal IgG Antibody.

Crystallization of IgG antibodies has important applications in the fields of structural biology, biotechnology, and biopharmaceutics. However, a rational approach to crystallize antibodies is still lacking. In this work, we report a method to estimate the solubility of antibodies at various temperatures. We experimentally determined the full phase diagram of an IgG antibody. Using the full diagram, we examined the metastability gaps, i.e., the distance between the crystal solubility line and the liquid-liquid coexistence curve, of IgG antibodies. By comparing our results to the partial phase diagrams of other IgGs reported in literature, we found that IgG antibodies have similar metastability gaps. Thereby, we present an equation with two phenomenological parameters to predict the approximate location of the solubility line of IgG antibodies with respect to their liquid-liquid coexistence curves. We have previously shown that the coexistence curve of an antibody solution can be readily determined by the polyethylene glycol-induced liquid-liquid phase separation method. Combining the polyethylene glycol-induced liquid-liquid phase separation measurements and the phenomenological equation in this article, we provide a general and practical means to predict the thermodynamic conditions for crystallizing IgG antibodies in the solution environments of interest.

Compartment-Specific Activation of the Proton-Sensor GPR65 is Uncoupled from Receptor Trafficking.

The canonical view of G protein-coupled receptor (GPCR) function is that receptor trafficking is tightly coupled to signaling. GPCRs remain on the plasma membrane (PM) at the cell surface until they are activated, after which they are desensitized and internalized into endosomal compartments. This canonical view presents an interesting context for proton-sensing GPCRs because they are more likely to be activated in acidic endosomal compartments than at the PM. Here we show that the trafficking of the prototypical proton-sensor GPR65 is fully uncoupled from signaling, unlike that of other known mammalian GPCRs. GPR65 internalized and localized to early and late endosomes, from where they signal at steady state, irrespective of extracellular pH. Acidic extracellular environments stimulated receptor signaling at the PM in a dose-dependent manner, although endosomal GPR65 was still required for a full signaling response. Receptor mutants that were incapable of activating cAMP trafficked normally, internalized, and localized to endosomal compartments. Our results show that GPR65 is constitutively active in endosomes, and suggest a model where changes in extracellular pH reprograms the spatial pattern of receptor signaling and biases the location of signaling to the cell surface.

Predictable cholesterol binding sites in GPCRs lack consensus motifs.

A rich diversity of transmembrane G protein-coupled receptors (GPCRs) are used by eukaryotes to sense physical and chemical signals. In humans alone, 800 GPCRs comprise the largest and most therapeutically targeted receptor class. Recent advances in GPCR structural biology have produced hundreds of GPCR structures solved by X-ray diffraction and increasingly, cryo-electron microscopy (cryo-EM). Many of these structures are stabilized by site-specific cholesterol binding, but it is unclear whether these interactions are a product of recurring cholesterol-binding motifs and if observed patterns of cholesterol binding differ by experimental technique. Here, we comprehensively analyze the location and composition of cholesterol binding sites in the current set of 473 human GPCR structural chains. Our findings establish that cholesterol binds similarly in cryo-EM and X-ray structures and show that 92% of cholesterol molecules on GPCR surfaces reside in predictable locations that lack discernable cholesterol-binding motifs.