Franziska Marie Heydenreich

G protein-coupled receptors (GPCRs) are important therapeutic drug targets for a wide range of diseases. Upon activation, GPCRs can initiate several signaling pathways, each with unique therapeutic implications. Therefore, understanding how drugs selectively engage specific signaling pathways becomes paramount. However, achieving this selectivity remains highly challenging. To unravel the underlying multifaceted mechanisms, we integrate systematic mutagenesis of the CB₂R, comprehensive profiling of G_αi2 and β-arrestin1 engagements and computer simulations to track the effects of mutations on receptor dynamics. Our research reveals multiple triggers within a complex allosteric communication network (ACN) that converge to preferential CB₂R coupling by modulating evolutionarily conserved motifs. Utilizing network path analysis, we find that potent triggers are typically highly connected nodes and are located near regions of high information transmission within the ACN. Our insights highlight the complexity of GPCR signaling and provide a framework for the rational design of drug candidates tailored to evoke specific functional responses, ultimately enhancing the precision and efficacy of therapeutic interventions.

Heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) bind to extracellular ligands and drugs and modulate intracellular responses through conformational changes. Despite their importance as drug targets, the molecular origins of pharmacological properties such as efficacy (maximum signaling response) and potency (the ligand concentration at half-maximal response) remain poorly understood for any ligand-receptor-signaling system. We used the prototypical adrenaline–β2 adrenergic receptor–G protein system to reveal how specific receptor residues decode and translate the information encoded in a ligand to mediate a signaling response. We present a data science framework to integrate pharmacological and structural data to uncover structural changes and allosteric networks relevant for ligand pharmacology. These methods can be tailored to study any ligand-receptor-signaling system, and the principles open possibilities for designing orthosteric and allosteric compounds with defined signaling properties.

Activation of the G protein-coupled receptors by agonists may result in the activation of one or more G proteins and recruitment of arrestins. The extent of the activation of each of these pathways depends on the intrinsic efficacy of the ligand. Quantification of intrinsic efficacy relative to a reference compound is essential for the development of novel compounds. In the operational model, changes in efficacy can be compensated by changes in the "functional" affinity, resulting in poorly defined values. To separate the effects of ligand affinity from the intrinsic activity of the receptor, we developed a Michaelis-Menten based quantification of G protein activation bias that uses experimentally measured ligand affinities and provides a single measure of ligand efficacy. We used it to evaluate the signalling of a promiscuous model receptor, the Vasopressin V2 receptor (V2R). Using BRET-based biosensors, we show that the V2R engages many different G proteins across all G protein subfamilies in response to its primary endogenous agonist, arginine vasopressin (AVP), including Gs and members of the Gi/o and G12/13 families. These signaling pathways are also activated by the synthetic peptide desmopressin, oxytocin, and the non-mammalian hormone vasotocin. We compared bias quantification using the operational model with Michaelis-Menten based quantification, the latter accurately quantified ligand efficacies despite large difference in ligand affinities. Together, these results showed that V2R is promiscuous in its ability to engage several G proteins and that its9 signaling profile is biased by small structural changes in the ligand. Significance Statement By modelling the G protein activation as Michaelis-Menten reaction, we developed a novel way of quantifying signalling bias. V2R activates or at least engages G proteins from all G protein subfamilies, including Gi2, Gz, Gq, G12, and G13. Their relative activation may explain its Gs-independent signalling.

G-protein-coupled receptors do not only feature the orthosteric pockets, where most endogenous agonists bind, but also a multitude of other allosteric pockets that have come into the focus as potential binding sites for synthetic modulators. Here, to better characterise such pockets, we investigate 557 GPCR structures by exhaustively docking small molecular probes in silico and converting the ensemble of binding locations to pocket-defining volumes. Our analysis confirms all previously identified pockets and reveals nine previously untargeted sites. In order to test for the feasibility of functional modulation of receptors through binding of a ligand to such sites, we mutate residues in two sites, in two model receptors, the muscarinic acetylcholine receptor M₃andβ₂-adrenergic receptor. Moreover, we analyse the correlation of inter-residue contacts with the activation states of receptors and show that contact patterns closely correlating with activation indeed coincide with these sites.

Two-thirds of human hormones and one-third of clinical drugs activate ~350 G-protein-coupled receptors (GPCR) belonging to four classes: A, B1, C and F. Whereas a model of activation has been described for class A, very little is known about the activation of the other classes, which differ by being activated by endogenous ligands bound mainly or entirely extracellularly. Here we show that, although they use the same structural scaffold and share several ‘helix macroswitches’, the GPCR classes differ in their ‘residue microswitch’ positions and contacts. We present molecular mechanistic maps of activation for each GPCR class and methods for contact analysis applicable for any functional determinants. This provides a superfamily residue-level rationale for conformational selection and allosteric communication by ligands and G proteins, laying the foundation for receptor-function studies and drugs with the desired modality.

Summary: Binding of arrestin to phosphorylated G protein-coupled receptors (GPCRs) is crucial for modulating signaling. Once internalized some GPCRs may complex with arrestin, while others interact transiently; this difference affects receptor signaling and recycling. Cell-based and in vitro biophysical assays reveal the role of membrane phosphoinositides (PIPs) in arrestin recruitment and GPCR-arrestin complex dynamics. We find that GPCRs broadly stratify into two groups, one requiring PIP-binding for arrestin recruitment and one that does not. Plasma membrane PIPs potentiate an active conformation of arrestin and stabilize GPCR-arrestin complexes by promoting a receptor core-engaged state of the complex. As allosteric modulators of GPCR-arrestin complex dynamics, membrane PIPs allow for additional conformational diversity beyond that imposed by GPCR phosphorylation alone. The dependance on membrane PIPs provides a mechanism for arrestin release from transiently associated GPCRs, allowing their rapid recycling, while explaining how stably associated GPCRs are able to engage G proteins at endosomes.

G protein-coupled receptors (GPCRs) translate binding of extracellular ligands into intracellular responses through conformational changes. Ligand properties are described by the maximum response (efficacy) and the agonist concentration at half-maximal response (potency). Integrating structural changes with pharmacological properties remains challenging and has not yet been performed at the resolution of individual amino acids. We use epinephrine and β2-adrenergic receptor as a model to integrate residue-level pharmacology data with intramolecular residue contact data describing receptor activation. This unveils the allosteric networks driving ligand efficacy and potency. We provide detailed insights into how structural rearrangements are linked to fundamental pharmacological properties at single-residue level in a receptor-ligand system. Our approach can be used to determine such pharmacological networks for any receptor-ligand complex.

G protein-coupled receptors do not only feature the orthosteric pockets, where most endogenous agonists bind, but also a multitude of other allosteric pockets that have come into the focus as potential binding sites for synthetic modulators. We have investigated 557 GPCR structures to better characterise such pockets by exhaustively docking small molecular probes in silico and converting the ensemble of binding locations to pocket-defining volumes. Our analysis confirmed all previously identified pockets and revealed nine previously untargeted sites. In order to test for the feasibility of functional modulation of receptors through binding of a ligand to such sites, we mutated residues in two sites in two model receptors. Moreover, we analysed the correlation of inter-residue contacts with the activation states of receptors and showed that contact patterns closely correlated with activation indeed coincide with these sites.

G protein coupled receptors (GPCRs) translate the actions of hormones into intracellular signalling events. Mutations in GPCRs can prevent their correct expression and trafficking to the cell surface and cause disease. We use single cell measurements in HEK293 cells to show that the balance between endoplasmic reticulum (ER) and cell surface localisation of the Vasopressin 2 receptor (V2R) varies significantly from cell to cell. We find that mutations in the V2R affect the proportion of cells able to send this GPCR to the cell surface but do not prevent all cells in the population from correctly trafficking the mutant receptors. These findings reveal that the ability of cells to correctly traffic V2R to the cell surface depends not only on the expressed V2R mutant but also on the individual cell environment. Significance statement: Missense mutations in the Vasopressin 2 Receptor (V2R) cause Nephrogenic Diabetes Insipidus. Some of these mutations prevent correct expression and trafficking of V2R to the cell surface resulting in a loss-of-function. We show -using single cell measurements-that the balance between endoplasmic reticulum and cell surface localisation of the V2R varies significantly from cell to cell, independent from its expression level. Mutations affect the proportion of cells able to send V2R to the cell surface but do not prevent all cells in the population from correctly trafficking the mutant receptors. Hence, the ability of cells to correctly traffic V2R to the cell surface depends not only on expressed V2R mutant but also on the cell environment.