G protein-coupled receptors (GPCRs) orchestrate diverse physiological responses via intracellular signaling through G proteins, GPCR kinases (GRKs), and arrestins. While the role of G proteins in receptor signaling is well-established, the contributions of GRKs and arrestins remain incompletely understood. Here, we investigate the influence of arrestin-interacting GPCR domains (helix-bundle/C-terminus) on β-arrestin conformations and functions using refined biosensors and advanced cellular knockout systems. By focusing on prototypical class A (b2AR) and B (V2R) receptors and their chimeras (b2V2/V2b2), we can now characterize differential β-arrestin conformational changes as primarily mediated by the receptor C-terminus or helix-bundle. Moreover, we demonstrate that some β-arrestin-supported processes are governed by distinct receptor domains, such as ERK1/2 activation (helix-bundle) and arrestin co-internalization (C-terminus), while others depend on the overall GPCR configuration, such as receptor internalization. Our findings elucidate how individual GPCR domains dictate downstream signaling events, shedding light on the structural basis of receptor-specific signaling and regulation. ### Competing Interest Statement S.S. is the founder and scientific advisor of 7TM Antibodies GmbH, Jena, Germany. The remaining authors declare no competing interests.

Abstract ID 98801Poster Board 159Dopamine receptors (DARs) are G-protein coupled receptors (GPCRs) that regulate diverse physiological functions including cognition, mood, movement, and reward-related behaviors. They are classified as either D1-like (D1R and D5R) or D2-like (D2R, D3R, and D4R) based on structural homology and pharmacological properties. The D1-like DARs couple to Gs and Golf to increase cAMP levels, while the D2-like DARs couple to Gi/o to decrease cAMP levels. All DARs also recruit β-arrestin which activates distinct signaling cascades and can also initiate receptor desensitization and internalization. There are seven GRK isoforms (GRKs1-7), with GRK2, GRK3, GRK5, and GRK6 being widely expressed. Using systematic mutational analyses, our lab has previously mapped the GRK-mediated phosphorylation sites on both the D1R and D2R, with phosphorylation occurring exclusively on ICL3 for the D2R, and both ICL3 and the C-terminus for the D1R. GRK-mediated phosphorylation of the D1R was found to be required for subsequent β-arrestin recruitment. In contrast, while GRKs play a role in β-arrestin recruitment to the D2R, GRK-mediated D2R phosphorylation is dispensable for this process. We have now sought to determine which GRK isoform(s) are involved in regulating β-arrestin recruitment to these receptors using cell lines in which the expression of specific GRK isoforms were selectively eliminated via CRISPR, as well as by utilizing isoform-selective GRK inhibitors. β-arrestin recruitment to both the D1R and D2R was severely impaired in cells lacking all GRKs (total KO), whereas selective expression of GRK isoforms 2, 3, 5 and 6 in the total KO cells was able to rescue β-arrestin recruitment. The kinase activity of each GRK was required for this recue for the D1R. Interestingly, the GRK2/3 and GRK5/6 isoforms differed in their requirement of kinase activity for the rescue of β-arrestin recruitment to the D2R. Individual and double GRK KO cells revealed that the GRK5/6 subfamily is more important for β-arrestin recruitment to the D1R, while the GRK2/3 subfamily is more important for the D2R. Treatment with GRK2/3-selective inhibitors recapitulated these findings with the GRK KO cells in that they impaired β-arrestin recruitment to the D2R, but had limited effect on this process for the D1R. Intriguingly, in cells with endogenous levels of GRK expression, β-arrestin recruitment to both the D1R and D2R were unaffected by inhibitors of GRK5/6. However, these inhibitors could block the rescue of β-arrestin recruitment to the D2R observed with GRK5 or GRK6 overexpression but had little effect on the rescue of β-arrestin recruitment to the D1R or the Gs-coupled β2 adrenergic receptor. As GRK distribution varies by tissue and brain region, it is intriguing to postulate that DAR regulation, and GPCR regulation in general, by specific GRK isoforms can add layers of regulatory fine-tuning through differentially directing signaling or trafficking outcomes.National Institute of Neurological Disorders and Stroke Intramural Research Program, National Institutes of Health (ZIA NS002263)

Sepsis is characterized as life-threatening organ dysfunction caused by a dysregulated host response to an infection. Despite numerous clinical trials that addressed this syndrome, there is still no causative treatment available to dampen its severity. Curtailing the infection at an early stage with anti-infectives is the only effective treatment regime besides intensive care. In search for additional treatment options, we recently discovered the inhibition of the sphingosine 1-phosphate (S1P) lyase and subsequent activation of the S1P receptor type 3 (S1PR3) in pre-conditioning experiments as promising targets for sepsis prevention. Here, we demonstrate that treatment of septic mice with the direct S1P lyase inhibitor C31 or the S1PR3 agonist CYM5541 in the advanced phase of sepsis resulted in a significantly increased survival rate. A single dose of each compound led to a rapid decline of sepsis severity in treated mice and coincided with decreased cytokine release and increased lung barrier function with unaltered bacterial load. The survival benefit of both compounds was completely lost in S1PR3 deficient mice. Treatment of the murine macrophage cell line J774.1 with either C31 or CYM5541 resulted in decreased protein kinase B (Akt) and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) phosphorylation without alteration of the mitogen-activated protein kinase (MAPK) p38 and p44/42 phosphorylation. Thus, activation of S1PR3 in the acute phase of sepsis by direct agonism or S1P lyase inhibition dampened Akt and JNK phosphorylation, resulting in decreased cytokine release, improved lung barrier stability, rapid decline of sepsis severity and better survival in mice.

Background: Bile salts of hepatic and microbial origin mediate interorgan cross talk in the gut-liver axis. Here, we assessed whether the newly discovered class of microbial bile salt conjugates (MBSCs) activate the main host bile salt receptors (Takeda G protein-coupled receptor 5 [TGR5] and farnesoid X receptor [FXR]) and enter the human systemic and enterohepatic circulation. Methods: N-amidates of (chenodeoxy) cholic acid and leucine, tyrosine, and phenylalanine were synthesized. Receptor activation was studied in cell-free and cell-based assays. MBSCs were quantified in mesenteric and portal blood and bile of patients undergoing pancreatic surgery. Results: MBSCs were activating ligands of TGR5 as evidenced by recruitment of Gsα protein, activation of a cAMP-driven reporter, and diminution of lipopolysaccharide-induced cytokine release from macrophages. Intestine-enriched and liver-enriched FXR isoforms were both activated by MBSCs, provided that a bile salt importer was present. The affinity of MBSCs for TGR5 and FXR was not superior to host-derived bile salt conjugates. Individual MBSCs were generally not detected (ie, < 2.5 nmol/L) in human mesenteric or portal blood, but Leu-variant and Phe-variant were readily measurable in bile, where MBSCs comprised up to 213 ppm of biliary bile salts. Conclusions: MBSCs activate the cell surface receptor TGR5 and the transcription factor FXR and are substrates for intestinal (apical sodium-dependent bile acid transporter) and hepatic (Na+ taurocholate co-transporting protein) transporters. Their entry into the human circulation is, however, nonsubstantial. Given low systemic levels and a surplus of other equipotent bile salt species, the studied MBSCs are unlikely to have an impact on enterohepatic TGR5/FXR signaling in humans. The origin and function of biliary MBSCs remain to be determined.

More than 30 years after their discovery, arrestins are recognised multiprotein scaffolds that play essential roles in G protein-coupled receptor (GPCR) regulation and signalling. Originally named for their capacity to hinder GPCR coupling to G proteins and facilitate receptor desensitisation, arrestins have emerged as key hubs for a myriad of other functions, including receptor internalisation and scaffolding of signalling complexes. Recent structural studies have started to provide snapshots of the complexes formed by GPCRs and arrestins, supporting a wealth of biochemical data delineating the molecular determinants of such interactions. Furthermore, biophysical techniques have also provided key information with regards to the basal and active conformations of arrestins, and how these are affected upon GPCR activation. Here, we review the most recent advances on our understanding of GPCR-arrestin complexes, from structure to interactions of arrestins with the lipid bilayer and other proteins. We also present an updated view on the development of tools to study the conformational flexibility of arrestins, with the potential to provide experimental data to describe the dynamic models of arrestin activation.

G protein-coupled receptors (GPCRs) constitute the largest family of transmembrane proteins and play a crucial role in regulating diverse cellular functions. They transmit their signaling via binding to intracellular signal transducers and effectors, such as G proteins, GPCR kinases, and β-arrestins. To influence specific GPCR signaling behaviors, β-arrestins recruit effectors to form larger signaling complexes. Intriguingly, they facilitate divergent functions for the binding to different receptors. Recent studies relying on advanced structural approaches, novel biosensors and interactome analyses bring us closer to understanding how this specificity is achieved. In this article, we share our hypothesis of how active GPCRs induce specific conformational rearrangements within β-arrestins to reveal distinct binding interfaces, enabling the recruitment of a subset of effectors to foster specialized signaling complexes. Furthermore, we discuss methods of how to comprehensively assess β-arrestin conformational states and present the current state of research regarding the functionality of these multifaceted scaffolding proteins.

ID 19401 Poster Board 148 The D2 dopamine receptor (D2R) interacts with and signals through both G proteins and ss-arrestins to regulate important physiological processes, such as movement, reward circuitry, emotion, and cognition. ß-arrestins are believed to interact with G protein-coupled receptors (GPCRs) at two interfaces: the receptor core and the phosphorylated C-terminal tail or intracellular loops. GPCR kinases (GRKs) are the primary drivers of GPCR phosphorylation, and for many receptors, this post-translational modification is indispensable for ß-arrestin recruitment. However, GRK-mediated receptor phosphorylation is not required for ß-arrestin recruitment to the D2R, and the role of GRKs in D2R-ß-arrestin interactions remains largely unexplored. In this study, we used GRK knockout cells engineered using CRISPR-Cas9 technology to determine if ß-arrestin recruitment to the D2R was GRK-dependent. Genetic elimination of all GRK expression significantly decreased, but did not totally eliminate, both ß-arrestin recruitment to the D2R and receptor internalization. However, these processes were rescued upon re-introduction of various GRK isoforms in the cells via transient transfection. Further, treatment with compound 101, a pharmacological inhibitor of GRK2/3 isoforms, dose-dependently decreased ß-arrestin recruitment and receptor internalization, highlighting the importance of these kinases for D2R-ß-arrestin interactions. Interestingly, these results were recapitulated using a phosphorylation-deficient (PO4-null) D2R mutant, emphasizing that GRKs enhance D2R-ß-arrestin interactions independently from receptor phosphorylation. As GRK2/3 are well known to interact with the ß-γ subunits of G proteins following heterotrimer dissociation, we investigated the role of G proteins in GRK functionality at the D2R. Inhibition of G protein signaling with pertussis toxin modestly decreased ß-arrestin recruitment to the D2R in cells expressing endogenous amounts of GRKs but had no effect when GRK2 was overexpressed. We also used a mutant GRK2 that is unable to bind to ß-γ G protein subunits (GRK2-R587Q) and found that ß-γ interactions were not necessary for this function of GRK2. Next, we investigated if the catalytic activity of GRK2 is required to enhance D2R-ß-arrestin interactions. Overexpression of a catalytically inactive GRK2 mutant (GRK2-K220R) did not enhance ß-arrestin recruitment or receptor internalization in cells expressing endogenous amounts of GRKs. However, when transfected into GRK-KO cells, GRK2-K220R rescued ß-arrestin recruitment and D2R internalization, albeit to a slightly lesser extent than WT GRK2. However, we found that GRK2-K220R is recruited to the D2R less efficiently than WT GRK2 in both parental and GRK-KO cells. Therefore, its lack of effect when overexpressed in parental cells could be due to competition from endogenous GRKs. Further, the reduced ability of GRK2-K220R to interact with the D2R could explain its reduced ability to drive ß-arrestin recruitment compared to WT GRK2. In summary, we demonstrate that GRKs, especially GRK2/3, are necessary for maximal agonist-stimulated D2R-ß-arrestin interactions, however, their kinase activities are not required. We hypothesize that GRK2 might act through a novel non-catalytic, scaffolding mechanism to enhance ß-arrestin recruitment to the D2R, highlighting the diversity with which GPCRs can interact with signal transduction proteins.

ID 15411 Poster Board 351 Dopamine receptors (DARs) are G protein-coupled receptors (GPCRs) that regulate diverse physiological functions including movement, mood, cognition, and motivation, and are involved in the pathology and treatment of numerous neuropsychiatric disorders. The D1-like DARs (D1R and D5R) activate adenylyl cyclase and increase cAMP levels, while D2-like DARs (D2R, D3R, D4R) inhibit adenylyl cyclase and decrease cAMP levels. Additionally, all DARs recruit β-arrestin, leading to separate signaling cascades and the initiation of receptor desensitization and internalization. β-arrestin recruitment to the D1R is closely linked to receptor phosphorylation, and the D1R has been shown to be phosphorylated by various kinases including protein kinase A (PKA), protein kinase C (PKC), and G protein-coupled receptor kinases (GRKs). The D1R contains 32 intracellular serine and threonine residues within the C-terminus and third intracellular loop (ICL3) that serve as potential phosphorylation sites. Using mutational analyses, we previously identified the PKA- and PKC-mediated phosphorylation sites and showed that GRK4 constitutively phosphorylates the receptor. We have now identified the D1R residues that are phosphorylated by GRKs in response to dopamine (DA) stimulation and found that mutation of these residues severely impairs β-arrestin recruitment, but has little effect on G protein-mediated signaling. Our results also suggest that most of the DA-induced GRK phosphorylation of the D1R occurs at residues T360 and S362 in the proximal C-terminus, and that these residues are primarily responsible for β-arrestin recruitment to the receptor. We next sought to determine which GRK subtypes are involved in DA-induced β-arrestin recruitment to the D1R. Using CRISPR-edited HEK293 cells in which all the endogenous GRKs were knocked out, we found that DA-induced β-arrestin recruitment to the D1R is severely impaired in the absence of GRKs, but can be rescued by exogenous GRK overexpression. The kinase activity of the GRKs appears to be necessary for this effect, as a catalytically inactive K220R GRK2 mutant is unable to rescue DA-induced β-arrestin recruitment to the D1R. Using individual or combinatorial GRK KO cells, we found that GRK2 and GRK3 play little to no role in mediating DA-stimulated β-arrestin recruitment to the D1R, which contrasts with diminished β-arrestin recruitment to the D2R in GRK2 KO cells. However, we found that DA-induced β-arrestin recruitment to the D1R was significantly impaired in GRK5/6 and GRK6 KO cells. We found similar effects using multiple GRK2/3-selective inhibitors, in that they had no effect on β-arrestin recruitment to the D1R, while they severely diminished DA-induced β-arrestin recruitment to the D2R. We are currently using GRK5/6-selective inhibitors to determine if pharmacological inhibition of GRK5/6 imitates GRK5/6 knockout. Taken together, these results suggest that GRK6 is prominently involved in DA-induced β-arrestin recruitment to the D1R, and that different GRK subtypes regulate β-arrestin recruitment to the D1R vs. the D2R. Because GRK subtype distribution varies by tissue and brain region, it is intriguing to postulate that D1R phosphorylation by different GRKs may add layers of regulatory fine-tuning through differentially directing D1R signaling or trafficking outcomes.

β-arrestins mediate regulatory processes for over 800 different G protein-coupled receptors (GPCRs) by adopting specific conformations that result from the geometry of the GPCR–β-arrestin complex. However, whether β-arrestin1 and 2 respond differently for binding to the same GPCR is still unknown. Employing GRK knockout cells and β-arrestins lacking the finger-loop-region, we show that the two isoforms prefer to associate with the active parathyroid hormone 1 receptor (PTH1R) in different complex configurations (“hanging” and “core”). Furthermore, the utilisation of advanced NanoLuc/FlAsH-based biosensors reveals distinct conformational signatures of β-arrestin1 and 2 when bound to active PTH1R (P-R*). Moreover, we assess β-arrestin conformational changes that are induced specifically by proximal and distal C-terminal phosphorylation and in the absence of GPCR kinases (GRKs) (R*). Here, we show differences between conformational changes that are induced by P-R* or R* receptor states and further disclose the impact of site-specific GPCR phosphorylation on arrestin-coupling and function.

The D2 dopamine receptor (D2R) regulates numerous CNS functions including movement, cognition, emotion, and reward circuitry. Agonist binding to the D2R promotes the activation of G protein-mediated signaling and the recruitment of β-arrestin to the receptor. Activated D2Rs are also phosphorylated by members of the G protein-coupled receptor kinase (GRK) family. GRK-mediated receptor phosphorylation is canonically thought to promote β-arrestin recruitment to GPCRs, however, previous research has shown that receptor phosphorylation is not required for β-arrestin recruitment to the D2R. Thus, the role of GRKs in D2R-β-arrestin interactions remains unclear. In the current study, we examined the relationship between GRKs and β-arrestin recruitment using GRK overexpression and GRK knockout cells engineered using CRISPR-Cas9 technology. Overexpression of individual GRK isoforms in parental HEK293 cells potentiated D2R-β-arrestin interactions to varying degrees depending on the isoform. Complete knockout of all GRK isoforms in HEK293 cells (GRK-KO) significantly abrogated, but did not eliminate, β-arrestin recruitment to the D2R. Further, knockout of individual GRK isoforms had variable effects on D2R-β-arrestin interactions ranging from no effect to significant inhibition. Expression of individual GRK isoforms in the GRK-KO cells rescued D2R-mediated β-arrestin recruitment to levels seen in the parental HEK293 cells. Overall, in these experiments, GRK2 seemed to exhibit the greatest modulatory effects on D2R-β-arrestin interactions. We next evaluated the role of receptor phosphorylation in GRK-mediated effects using a phosphorylation-deficient (PO4-null) D2R mutant (Namkung et. al., JBC 284:34103, 2009). The WT-D2R and PO4-null-D2R exhibited identical levels of agonist-stimulated β-arrestin interactions as well as receptor internalization. Notably, inhibition of GRK catalytic activity attenuated agonist-stimulated β-arrestin recruitment to both the WT-D2R and PO4-null-D2R, suggesting the existence of an unknown GRK substrate. Upon recruitment to GPCRs, β-arrestins undergo conformational changes that correlate with their downstream signaling effects. Consequently, we sought to investigate if GRKs play a role in β-arrestin conformational changes seen upon D2R activation. To probe these phenomena, we used intramolecular fluorescent arsenical hairpin (FlAsH) nanoBRET β-arrestin biosensors. We found that GRK2 increased β-arrestin conformational changes induced by the WT-D2R and PO4-null-D2R. Further, activation of the PO4-null-D2R induced greater conformational changes in β-arrestin than with the WT-D2R, suggesting that receptor phosphorylation does not mediate β-arrestin activation by this GPCR. Taken together, these data suggest that GRKs function noncanonically to modulate D2R-β-arrestin interactions and highlight the need to identify additional GRK substrates to better inform our understanding of GPCR signaling.

G protein-coupled receptors (GPCRs) activate G proteins and undergo a complex regulation by interaction with GPCR kinases (GRKs) and the formation of receptor–arrestin complexes. However, the impact of individual GRKs on arrestin binding is not clear. We report the creation of eleven combinatorial HEK293 knockout cell clones lacking GRK2/3/5/6, including single, double, triple and the quadruple GRK knockout. Analysis of β-arrestin1/2 interactions for twelve GPCRs in our GRK knockout cells enables the differentiation of two main receptor subsets: GRK2/3-regulated and GRK2/3/5/6-regulated receptors. Furthermore, we identify GPCRs that interact with β-arrestins via the overexpression of specific GRKs even in the absence of agonists. Finally, using GRK knockout cells, PKC inhibitors and β-arrestin mutants, we present evidence for differential receptor–β-arrestin1/2 complex configurations mediated by selective engagement of kinases. We anticipate our GRK knockout platform to facilitate the elucidation of previously unappreciated details of GRK-specific GPCR regulation and β-arrestin complex formation.

Dopamine (DA) receptors (DARs) are G protein-coupled receptors (GPCRs) that regulate diverse physiological functions including movement, cognition, mood, and reward-related behaviors, as well as cardiovascular and renal physiology. Multiple diseases are linked to dysregulated dopaminergic functioning including Parkinson's disease, schizophrenia, substance use disorder, and hypertension. DARs are classified as either D1-like (D1R and D5R) or D2-like (D2R, D3R, and D4R) based on structural homology and pharmacological properties. The D1-like DARs (D1R and D5R) increase cAMP, while the D2-like DARs (D2R, D3R, D4R) decrease cAMP. All DARs also recruit β-arrestin which activates separate signaling cascades and also initiates receptor desensitization and internalization. Generally, agonist activation of GPCRs, including DARs, rapidly leads to receptor desensitization and a return to basal levels of signaling. This occurs even in the continued presence of agonist, ensuring homeostasis. This desensitization process is intimately linked with receptor phosphorylation. The D1R is highly phosphorylated, with 32 intracellular serine and threonine residues, and is known to be phosphorylated by several kinases including protein kinase A (PKA), protein kinase C (PKC), and G protein-coupled receptor kinases (GRKs). Previous studies by our lab indicate that the D1R is phosphorylated on its third intracellular loop (ICL3) and C-terminus in a hierarchical fashion, in that phosphorylation must first occur on the C-terminus before the ICL3 can be phosphorylated. Using systematic mutational analyses, we previously identified the PKC-mediated D1R phosphorylation sites. We have now extended these studies to completely identify the DA-induced, GRK-mediated phosphorylation sites on the D1R. We found that GRK-mediated phosphorylation involves several serine and threonine residues on the C-terminus and ICL3. Mutation of these residues to alanine or valine, respectively, abolishes DA-induced D1R phosphorylation and severely impairs β-arrestin recruitment, but causes little effect on G protein-mediated signaling. Our results indicate that a large fraction of DA-induced D1R phosphorylation occurs on residues T360 and S362 in the proximal C-terminus, and that these residues are also responsible for the majority of DA-induced β-arrestin recruitment to the D1R. Using HEK cells that have had their endogenous GRKs knocked out via CRISPR, we found that DA-induced β-arrestin recruitment to the D1R is severely impaired. However, β-arrestin recruitment can be restored by expressing exogenous GRKs in these cells, further suggesting that DA-induced β-arrestin recruitment to the D1R is highly dependent on GRK phosphorylation. As GRK distribution varies by tissue and brain region, it is intriguing to postulate that D1R phosphorylation by different GRKs can add layers of regulatory fine-tuning through differential effects on D1R signaling or trafficking outcomes.

G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane receptors and represent major drug targets. Upon ligand stimulation, GPCRs activate G proteins and undergo a complex regulation by interaction with GPCR kinases (GRKs) and formation of receptor–arrestin complexes. For many GPCRs, this mechanism triggers receptor desensitisation, internalisation, and possibly a second intracellular signalling wave. Here we created eleven different HEK293 knockout cell clones for GRK2, 3, 5, and 6 individually and in combination. These include four single, two double, four triple, and the quadruple GRK knockout. The statistical evaluation of β-arrestin1/2 interactions for twelve different receptors grouped the tested GPCRs into two main subsets: those for which β-arrestin interaction was mediated by either GRK2, 3, 5, or 6 and those that are mediated by GRK2 or 3 only. Interestingly, the overexpression of specific GRKs was found to induce a robust, ligand-independent β-arrestin interaction with the V2R and AT1R. Finally, using GRK knockout cells, PKC inhibitors, and β-arrestin mutants, we present evidence for differential AT1R–β-arrestin2 complex configurations mediated by selective engagement of PKC, GRK2, or GRK6. We anticipate our novel GRK-knockout platform to facilitate the elucidation of previously unappreciated details of GRK-specific GPCR regulation and β-arrestin complex formation.

G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane receptors and their signal transduction is tightly regulated by GPCR kinases (GRKs) and β-arrestins. In this review, we discuss novel aspects of the regulatory GRK/β-arrestin system. Therefore, we briefly revise the origin of the “barcode” hypothesis for GPCR/β-arrestin interactions, which states that β-arrestins recognize different receptor phosphorylation states to induce specific functions. We emphasize two important parameters which may influence resulting GPCR phosphorylation patterns: (A) direct GPCR–GRK interactions and (B) tissue-specific expression and availability of GRKs and β-arrestins. In most studies that focus on the molecular mechanisms of GPCR regulation, these expression profiles are underappreciated. Hence we analyzed expression data for GRKs and β-arrestins in 61 tissues annotated in the Human Protein Atlas. We present our analysis in the context of pathophysiological dysregulation of the GPCR/GRK/β-arrestin system. This tissue-specific point of view might be the key to unraveling the individual impact of different GRK isoforms on GPCR regulation.