Optimization of a Glucagon-Like Peptide 1 Receptor Antagonist Antibody for Treatment of Hyperinsulinism.

Congenital hyperinsulinism (HI) is a genetic disorder in which pancreatic ß-cell insulin secretion is excessive and results in hypoglycemia that, without treatment, can cause brain damage or death. Most patients with loss-of-function mutations in ABCC8 and KCNJ11, the genes encoding the ß-cell ATP-sensitive potassium channel (KATP), are unresponsive to diazoxide, the only U.S. Food and Drug Administration-approved medical therapy and require pancreatectomy. The glucagon-like peptide 1 receptor (GLP-1R) antagonist exendin-(9-39) is an effective therapeutic agent that inhibits insulin secretion in both HI and acquired hyperinsulinism. Previously, we identified a highly potent antagonist antibody, TB-001-003, which was derived from our synthetic antibody libraries that were designed to target G protein-coupled receptors. Here, we designed a combinatorial variant antibody library to optimize the activity of TB-001-003 against GLP-1R and performed phage display on cells overexpressing GLP-1R. One antagonist, TB-222-023, is more potent than exendin-(9-39), also known as avexitide. TB-222-023 effectively decreased insulin secretion in primary isolated pancreatic islets from a mouse model of hyperinsulinism, Sur1-/- mice, and in islets from an infant with HI, and increased plasma glucose levels and decreased the insulin to glucose ratio in Sur1-/- mice. These findings demonstrate that targeting GLP-1R with an antibody antagonist is an effective and innovative strategy for treatment of hyperinsulinism. ARTICLE HIGHLIGHTS: Patients with the most common and severe form of diazoxide-unresponsive congenital hyperinsulinism (HI) require a pancreatectomy. Other second-line therapies are limited in their use because of severe side effects and short half-lives. Therefore, there is a critical need for better therapies. Studies with the glucagon-like peptide 1 receptor (GLP-1R) antagonist, avexitide (exendin-(9-39)), have demonstrated that GLP-1R antagonism is effective at lowering insulin secretion and increasing plasma glucose levels. We have optimized a GLP-1R antagonist antibody with more potent blocking of GLP-1R than avexitide. This antibody therapy is a potential novel and effective treatment for HI.

Discovery and design of G protein-coupled receptor targeting antibodies.

INTRODUCTION: G protein-coupled receptors (GPCRs) are the target of one-third of all approved drugs; however, these drugs only target about one-eighth of the human repertoire of GPCRs. GPCRs regulate a diverse range of critical physiological processes including organ development, cardiovascular function, mood, cognition, multicellularity, cellular motility, immune responses and sensation of light, taste, and odor. However, many GPCRs are expressed poorly, and a significant proportion have unknown ligands and unclear signaling pathways. AREAS COVERED: GPCRs are better suited to be targeted by monoclonal antibodies (mAbs) because of the challenges encountered in small-molecule discoveries such as druggability, selectivity, and distribution. mAbs have better drug-like properties in these respects. Herein, the authors review previously discovered functional mAbs that target GPCRs that are in the clinic and/or in development. They also review the biophysical considerations that make GPCRs so challenging to work with but also provide opportunities for biologic druggability. EXPERT OPINION: GPCRs are proven targets of small molecules yet remain an under-represented target of biologics. We believe that antibody drugs that target GPCRs have the potential to unlock new therapeutic avenues and also uncover previously unappreciated receptor biology, particularly when harnessing next-generation biologic modalities.

Stability of patch-turnover relationships under equilibrium and nonequilibrium metapopulation dynamics driven by biogeography.

Two controversial tenets of metapopulation biology are whether patch quality and the surrounding matrix are more important to turnover (colonisation and extinction) than biogeography (patch area and isolation) and whether factors governing turnover during equilibrium also dominate nonequilibrium dynamics. We tested both tenets using 18 years of surveys for two secretive wetland birds, black and Virginia rails, during (1) a period of equilibrium with stable occupancy and (2) after drought and arrival of West Nile Virus (WNV), which resulted in WNV infections in rails, increased extinction and decreased colonisation probabilities modified by WNV, nonequilibrium dynamics for both species and occupancy decline for black rails. Area (primarily) and isolation (secondarily) drove turnover during both stable and unstable metapopulation dynamics, greatly exceeding the effects of patch quality and matrix conditions. Moreover, slopes between turnover and patch characteristics changed little between equilibrium and nonequilibrium, confirming the overriding influences of biogeographic factors on turnover.

Receptor Interactome Discovery with RDIMIS, a Membrane Protein Interaction Screen Using Recombinant Extracellular Vesicles.

Membrane protein interactions are challenging to identify because of the unique biophysical characteristics of both transmembrane proteins and membrane environments. The Receptor Display in Membranes Interaction Screen (RDIMIS) platform overcomes these challenges by screening transmembrane and membrane-proximal proteins in a membrane environment using recombinant extracellular vesicles (rEVs). The screen has been used to successfully identify interactions for difficult-to-study receptors in an unbiased manner. In this report, we detail how we generate rEVs, characterize the rEVs to ensure screen-readiness, and perform the full interaction screening, with emphasis on the criteria necessary to obtain clear, interpretable results. We also include support protocols for generating a screening library and validating screening results, as well as an alternate protocol for RDIMIS enabling the profiling of naturally occurring extracellular vesicles. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Generating and isolating extracellular vesicles from cells Basic Protocol 2: Characterizing recombinant extracellular vesicles Support Protocol 1: Preparing the receptor screening library Basic Protocol 3: Performing the Receptor Display in Membranes Interaction Screen (RDIMIS) Support Protocol 2: Validating RDIMIS results using microscopy Alternate Protocol: Detecting unlabeled endogenous vesicles.

The neutrophil protein CD177 is a novel PDPN receptor that regulates human cancer-associated fibroblast physiology.

The cancer-associated fibroblast (CAF) marker podoplanin (PDPN) is generally correlated with poor clinical outcomes in cancer patients and thus represents a promising therapeutic target. Despite its biomedical relevance, basic aspects of PDPN biology such as its cellular functions and cell surface ligands remain poorly uncharacterized, thus challenging drug development. Here, we utilize a high throughput platform to elucidate the PDPN cell surface interactome, and uncover the neutrophil protein CD177 as a new binding partner. Quantitative proteomics analysis of the CAF phosphoproteome reveals a role for PDPN in cell signaling, growth and actomyosin contractility, among other processes. Moreover, cellular assays demonstrate that CD177 is a functional antagonist, recapitulating the phenotype observed in PDPN-deficient CAFs. In sum, starting from the unbiased elucidation of the PDPN co-receptome, our work provides insights into PDPN functions and reveals the PDPN/CD177 axis as a possible modulator of fibroblast physiology in the tumor microenvironment.

A membrane protein display platform for receptor interactome discovery.

Cell surface receptors are critical for cell signaling and constitute a quarter of all human genes. Despite their importance and abundance, receptor interaction networks remain understudied because of difficulties associated with maintaining membrane proteins in their native conformation and their typically weak interactions. To overcome these challenges, we developed an extracellular vesicle-based method for membrane protein display that enables purification-free and high-throughput detection of receptor-ligand interactions in membranes. We demonstrate that this platform is broadly applicable to a variety of membrane proteins, enabling enhanced detection of extracellular interactions over a wide range of binding affinities. We were able to recapitulate and expand the interactome for prominent members of the B7 family of immunoregulatory proteins such as PD-L1/CD274 and B7-H3/CD276. Moreover, when applied to the orphan cancer-associated fibroblast protein, LRRC15, we identified a membrane-dependent interaction with the tumor stroma marker TEM1/CD248. Furthermore, this platform enabled profiling of cellular receptors for target-expressing as well as endogenous extracellular vesicles. Overall, this study presents a sensitive and easy to use screening platform that bypasses membrane protein purification and enables characterization of interactomes for any cell surface-expressed target of interest in its native state.

Correction: Evaluating outcomes of management targeting the recovery of a migratory songbird of conservation concern.

[This corrects the article DOI: 10.7717/peerj.4319.].

The dopamine D2 receptor can directly recruit and activate GRK2 without G protein activation.

The dopamine D2 receptor (D2R) is a G protein-coupled receptor (GPCR) that is critical for many central nervous system functions. The D2R carries out these functions by signaling through two transducers: G proteins and ß-arrestins (ßarrs). Selectively engaging either the G protein or ßarr pathway may be a way to improve drugs targeting GPCRs. The current model of GPCR signal transduction posits a chain of events where G protein activation ultimately leads to ßarr recruitment. GPCR kinases (GRKs), which are regulated by G proteins and whose kinase action facilitates ßarr recruitment, bridge these pathways. Therefore, ßarr recruitment appears to be intimately tied to G protein activation via GRKs. Here we sought to understand how GRK2 action at the D2R would be disrupted when G protein activation is eliminated and the effect of this on ßarr recruitment. We used two recently developed biased D2R mutants that can preferentially interact either with G proteins or ßarrs as well as a ßarr-biased D2R ligand, UNC9994. With these functionally selective tools, we investigated the mechanism whereby the ßarr-preferring D2R achieves ßarr pathway activation in the complete absence of G protein activation. We describe how direct, G protein-independent recruitment of GRK2 drives interactions at the ßarr-preferring D2R and also contributes to ßarr recruitment at the WT D2R. Additionally, we found an additive interaction between the ßarr-preferring D2R mutant and UNC9994. These results reveal that the D2R can directly recruit GRK2 without G protein activation and that this mechanism may have relevance to achieving ßarr-biased signaling.

Population trends in Vermivora warblers are linked to strong migratory connectivity.

Migratory species can experience limiting factors at different locations and during different periods of their annual cycle. In migratory birds, these factors may even occur in different hemispheres. Therefore, identifying the distribution of populations throughout their annual cycle (i.e., migratory connectivity) can reveal the complex ecological and evolutionary relationships that link species and ecosystems across the globe and illuminate where and how limiting factors influence population trends. A growing body of literature continues to identify species that exhibit weak connectivity wherein individuals from distinct breeding areas co-occur during the nonbreeding period. A detailed account of a broadly distributed species exhibiting strong migratory connectivity in which nonbreeding isolation of populations is associated with differential population trends remains undescribed. Here, we present a range-wide assessment of the nonbreeding distribution and migratory connectivity of two broadly dispersed Nearctic-Neotropical migratory songbirds. We used geolocators to track the movements of 70 Vermivora warblers from sites spanning their breeding distribution in eastern North America and identified links between breeding populations and nonbreeding areas. Unlike blue-winged warblers (Vermivora cyanoptera), breeding populations of golden-winged warblers (Vermivora chrysoptera) exhibited strong migratory connectivity, which was associated with historical trends in breeding populations: stable for populations that winter in Central America and declining for those that winter in northern South America.

Evaluating outcomes of management targeting the recovery of a migratory songbird of conservation concern.

BACKGROUND: Assessing outcomes of habitat management is critical for informing and adapting conservation plans. From 2013-2019, a multi-stage management initiative, led by the American Bird Conservancy (ABC), aims to create >25,000 ha of shrubland and early-successional vegetation to benefit Golden-winged Warblers (Vermivora chrysoptera) in managed forested landscapes of the western Great Lakes region. We studied a dense breeding population of Golden-winged Warblers at Rice Lake National Wildlife Refuge (NWR) in Minnesota, USA, where ABC initiative management was implemented to benefit the species. METHODS: We monitored abundance before (2011-2014) and after (2015-2016) management, and we estimated full-season productivity (i.e., young recruited into the fall population) from predictive, spatially explicit models, informed by nest and fledgling survival data collected at sites in the western Great Lakes region, including Rice Lake NWR, during 2011 and 2012. Then, using biologically informed models of bird response to observed and predicted vegetation succession, we estimated the cumulative change in population recruitment over various scenarios of vegetation succession and demographic response. RESULTS: We observed an 32% decline in abundance of breeding pairs and estimated a 27% decline in per-pair full-season productivity following management, compared to no change in a nearby control site. In models that ranged from highly optimistic to progressively more realistic scenarios, we estimated a net loss of 72-460 juvenile Golden-winged Warblers produced from the managed site in the 10-20 years following management. Even if our well-informed and locally validated productivity models produced erroneous estimates and the management resulted in only a temporary reduction in abundance (i.e., no change in productivity), our forecast models still predicted a net loss of 137-260 juvenile Golden-winged Warblers from the managed area over the same time frame. CONCLUSIONS: Our study site represents only a small portion of a massive management initiative; however, the management at our site was conducted in accordance with the initiative's management plans, the resulting vegetation structure is consistent with that of other areas managed under the initiative, and those responsible for the initiative have described the management at our study site as successful Golden-winged Warbler management. Our assessment demonstrates that, at least for the only site for which pre- and post-management data on Golden-winged Warblers exist, the ABC management initiative is having a substantial and likely enduring negative impact on the species it purports to benefit. We suggest that incorporating region-specific, empirical information about Golden-winged Warbler-habitat relations into habitat management efforts would increase the likelihood of a positive response by Golden-winged Warblers.

Response to Lisovski et al.

Lisovski et al.[1] describe the widely recognized limitations of light-level geolocator data for identifying short-distance latitudinal movements, recommend that caution be used when interpreting such data, intimated that we did not use such caution and argued that environmental shading likely explained the Golden-winged Warbler (Vermivora chrysoptera) movements described in our 2015 report [2]. Lisovski et al.[1] conclude that the bird movements we reported could not be disentangled from estimation error in stationary animals caused by environmental shading. We argue that, to the contrary, these hypotheses can easily be disentangled because the premise that environmental shading caused synchronous and parallel error among geolocators is false. With their assertion that our location estimates could be biased by >3,500 km on a day with no observable local sources of shading, Lisovski et al.[1] have taken a position of incredulity toward all geolocator-based animal movement data published to date.

Engineered D2R Variants Reveal the Balanced and Biased Contributions of G-Protein and ß-Arrestin to Dopamine-Dependent Functions.

The dopamine D2 receptor (D2R), like many G-protein-coupled receptors, signals through G-protein- and ß-arrestin-dependent pathways. Preferential activation of one of these pathways is termed functional selectivity or biased signaling and is a promising therapeutic strategy. Though biased signaling through D2Rs has been demonstrated, acquiring the mechanistic details of biased D2R/G-protein and D2R/ß-arrestin signaling in vivo has been challenging because of the lack of techniques that specifically target these interactions in discrete cell populations. To address this question, we employed a cell type-specific viral expression approach to restore D2R variants that preferentially engage either G-protein or ß-arrestin signaling in 'indirect pathway' medium spiny neurons (iMSNs), because of their central role in dopamine circuitry. We found that the effect of haloperidol antagonism on D2R metabolic signaling events is largely mediated by acute blockade of D2R/G-protein signaling. We show that a D2R-driven behavior, nestlet shredding, is similarly driven by D2R/G-protein signaling. On the other hand, D2R-driven locomotion and rearing require coordinated D2R/G-protein and D2R/ß-arrestin signaling. The acute locomotor response to amphetamine and cocaine similarly depend on both G-protein and ß-arrestin D2R signaling. Surprisingly, another psychotropic drug, phencyclidine, displayed a selective D2R/ß-arrestin potentiation of locomotion. These findings highlight how D2R mostly relies upon balanced G-protein and ß-arrestin signaling in iMSNs. However, the response to haloperidol and phencyclidine indicates that normal D2R signaling homeostasis can be dramatically altered, indicating that targeting a specific D2R signal transduction pathway could allow for more precise modulation of dopamine circuit function.

New Concepts in Dopamine D2 Receptor Biased Signaling and Implications for Schizophrenia Therapy.

The dopamine D2 receptor (D2R) is a G protein-coupled receptor that is a common target for antipsychotic drugs. Antagonism of D2R signaling in the striatum is thought to be the primary mode of action of antipsychotic drugs in alleviating psychotic symptoms. However, antipsychotic drugs are not clinically effective at reversing cortical-related symptoms, such as cognitive deficits in schizophrenia. While the exact mechanistic underpinnings of these cognitive deficits are largely unknown, deficits in cortical dopamine function likely play a contributing role. It is now recognized that similar to most G protein-coupled receptors, D2Rs signal not only through canonical G protein pathways but also through noncanonical beta-arrestin2-dependent pathways. We review the current mechanistic bases for this dual signaling mode of D2Rs and how these new concepts might be leveraged for therapeutic gain to target both cortical and striatal dysfunction in dopamine neurotransmission and hence have the potential to correct both positive and cognitive symptoms of schizophrenia.

Molecular basis for multimerization in the activation of the epidermal growth factor receptor.

The epidermal growth factor receptor (EGFR) is activated by dimerization, but activation also generates higher-order multimers, whose nature and function are poorly understood. We have characterized ligand-induced dimerization and multimerization of EGFR using single-molecule analysis, and show that multimerization can be blocked by mutations in a specific region of Domain IV of the extracellular module. These mutations reduce autophosphorylation of the C-terminal tail of EGFR and attenuate phosphorylation of phosphatidyl inositol 3-kinase, which is recruited by EGFR. The catalytic activity of EGFR is switched on through allosteric activation of one kinase domain by another, and we show that if this is restricted to dimers, then sites in the tail that are proximal to the kinase domain are phosphorylated in only one subunit. We propose a structural model for EGFR multimerization through self-association of ligand-bound dimers, in which the majority of kinase domains are activated cooperatively, thereby boosting tail phosphorylation.

Receptor, Ligand and Transducer Contributions to Dopamine D2 Receptor Functional Selectivity.

Functional selectivity (or biased agonism) is a property exhibited by some G protein-coupled receptor (GPCR) ligands, which results in the modulation of a subset of a receptor's signaling capabilities and more precise control over complex biological processes. The dopamine D2 receptor (D2R) exhibits pleiotropic responses to the biogenic amine dopamine (DA) to mediate complex central nervous system functions through activation of G proteins and ß-arrestins. D2R is a prominent therapeutic target for psychological and neurological disorders in which DA biology is dysregulated and targeting D2R with functionally selective drugs could provide a means by which pharmacotherapies could be developed. However, factors that determine GPCR functional selectivity in vivo may be multiple with receptors, ligands and transducers contributing to the process. We have recently described a mutagenesis approach to engineer biased D2R mutants in which G protein-dependent ([Gprot]D2R) and ß-arrestin-dependent signaling ([ßarr]D2R) were successfully separated (Peterson, et al. PNAS, 2015). Here, permutations of these mutants were used to identify critical determinants of the D2R signaling complex that impart signaling bias in response to the natural or synthetic ligands. Critical residues identified in generating [Gprot]D2R and [ßarr]D2R conferred control of partial agonism at G protein and/or ß-arrestin activity. Another set of mutations that result in G protein bias was identified that demonstrated that full agonists can impart unique activation patterns, and provided further credence to the concept of ligand texture. Finally, the contributions and interplay between different transducers indicated that G proteins are not aberrantly activated, and that receptor kinase and ß-arrestin activities are inextricably linked. These data provide a thorough elucidation of the feasibility and malleability of D2R functional selectivity and point to means by which novel in vivo therapies could be modeled.

Elucidation of G-protein and ß-arrestin functional selectivity at the dopamine D2 receptor.

The neuromodulator dopamine signals through the dopamine D2 receptor (D2R) to modulate central nervous system functions through diverse signal transduction pathways. D2R is a prominent target for drug treatments in disorders where dopamine function is aberrant, such as schizophrenia. D2R signals through distinct G-protein and ß-arrestin pathways, and drugs that are functionally selective for these pathways could have improved therapeutic potential. How D2R signals through the two pathways is still not well defined, and efforts to elucidate these pathways have been hampered by the lack of adequate tools for assessing the contribution of each pathway independently. To address this, Evolutionary Trace was used to produce D2R mutants with strongly biased signal transduction for either the G-protein or ß-arrestin interactions. These mutants were used to resolve the role of G proteins and ß-arrestins in D2R signaling assays. The results show that D2R interactions with the two downstream effectors are dissociable and that G-protein signaling accounts for D2R canonical MAP kinase signaling cascade activation, whereas ß-arrestin only activates elements of this cascade under certain conditions. Nevertheless, when expressed in mice in GABAergic medium spiny neurons of the striatum, the ß-arrestin-biased D2R caused a significant potentiation of amphetamine-induced locomotion, whereas the G protein-biased D2R had minimal effects. The mutant receptors generated here provide a molecular tool set that should enable a better definition of the individual roles of G-protein and ß-arrestin signaling pathways in D2R pharmacology, neurobiology, and associated pathologies.

Targeting ß-arrestin2 in the treatment of L-DOPA-induced dyskinesia in Parkinson's disease.

Parkinson's disease (PD) is characterized by severe locomotor deficits and is commonly treated with the dopamine (DA) precursor l-3,4-dihydroxyphenylalanine (L-DOPA), but its prolonged use causes dyskinesias referred to as L-DOPA-induced dyskinesias (LIDs). Recent studies in animal models of PD have suggested that dyskinesias are associated with the overactivation of G protein-mediated signaling through DA receptors. ß-Arrestins desensitize G protein signaling at DA receptors (D1R and D2R) in addition to activating their own G protein-independent signaling events, which have been shown to mediate locomotion. Therefore, targeting ß-arrestins in PD L-DOPA therapy might prove to be a desirable approach. Here we show in a bilateral DA-depletion mouse model of Parkinson's symptoms that genetic deletion of ß-arrestin2 significantly limits the beneficial locomotor effects while markedly enhancing the dyskinesia-like effects of acute or chronic L-DOPA treatment. Viral rescue or overexpression of ß-arrestin2 in knockout or control mice either reverses or protects against LIDs and its key biochemical markers. In other more conventional animal models of DA neuron loss and PD, such as 6-hydroxydopamine-treated mice or rats and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated nonhuman primates, ß-arrestin2 overexpression significantly reduced dyskinesias while maintaining the therapeutic effect of L-DOPA. Considerable efforts are being spent in the pharmaceutical industry to identify therapeutic approaches to block LIDs in patients with PD. Our results point to a potential therapeutic approach, whereby development of either a genetic or pharmacological intervention to enhance ß-arrestin2- or limit G protein-dependent D1/D2R signaling could represent a more mechanistically informed strategy.

Tornadic storm avoidance behavior in breeding songbirds.

Migration is a common behavior used by animals of many taxa to occupy different habitats during different periods. Migrant birds are categorized as either facultative (i.e., those that are forced to migrate by some proximal cue, often weather) or obligate (i.e., those that migrate on a regular cycle). During migration, obligate migrants can curtail or delay flights in response to inclement weather or until favorable winds prevail, and they can temporarily reorient or reverse direction when ecological or meteorological obstacles are encountered. However, it is not known whether obligate migrants undertake facultative migrations and make large-scale movements in response to proximal cues outside of their regular migration periods. Here, we present the first documentation of obligate long-distance migrant birds undertaking a facultative migration, wherein breeding golden-winged warblers (Vermivora chrysoptera) carrying light-level geolocators performed a >1,500 km 5-day circumvention of a severe tornadic storm. The birds evacuated their breeding territories >24 hr before the arrival of the storm and atmospheric variation associated with it. The probable cue, radiating >1,000 km from tornadic storms, perceived by birds and influencing bird behavior and movements, is infrasound (i.e., sound below the range of human hearing). With the predicted increase in severity and frequency of similar storms as anthropogenic climate change progresses, understanding large-scale behavioral responses of animals to such events will be an important objective of future research.

G Protein and ß-arrestin signaling bias at the ghrelin receptor.

The G protein-coupled ghrelin receptor GHSR1a is a potential pharmacological target for treating obesity and addiction because of the critical role ghrelin plays in energy homeostasis and dopamine-dependent reward. GHSR1a enhances growth hormone release, appetite, and dopamine signaling through G(q/11), G(i/o), and G(12/13) as well as ß-arrestin-based scaffolds. However, the contribution of individual G protein and ß-arrestin pathways to the diverse physiological responses mediated by ghrelin remains unknown. To characterize whether a signaling bias occurs for GHSR1a, we investigated ghrelin signaling in a number of cell-based assays, including Ca(2+) mobilization, serum response factor response element, stress fiber formation, ERK1/2 phosphorylation, and ß-arrestin translocation, utilizing intracellular second loop and C-tail mutants of GHSR1a. We observed that GHSR1a and ß-arrestin rapidly form metastable plasma membrane complexes following exposure to an agonist, but replacement of the GHSR1a C-tail by the tail of the vasopressin 2 receptor greatly stabilizes them, producing complexes observable on the plasma membrane and also in endocytic vesicles. Mutations of the contiguous conserved amino acids Pro-148 and Leu-149 in the GHSR1a intracellular second loop generate receptors with a strong bias to G protein and ß-arrestin, respectively, supporting a role for conformation-dependent signaling bias in the wild-type receptor. Our results demonstrate more balance in GHSR1a-mediated ERK signaling from G proteins and ß-arrestin but uncover an important role for ß-arrestin in RhoA activation and stress fiber formation. These findings suggest an avenue for modulating drug abuse-associated changes in synaptic plasticity via GHSR1a and indicate the development of GHSR1a-biased ligands as a promising strategy for selectively targeting downstream signaling events.