The role of G protein-coupled receptor kinases in GLP-1R ß-arrestin recruitment and internalisation.

The glucagon-like peptide 1 receptor (GLP-1R) is a validated clinical target for the treatment of type 2 diabetes and obesity. Unlike most G protein-coupled receptors (GPCRs), the GLP-1R undergoes an atypical mode of internalisation that does not require ß-arrestins. While differences in GLP-1R trafficking and ß-arrestin recruitment have been observed between clinically used GLP-1R agonists, the role of G protein-coupled receptor kinases (GRKs) in affecting these pathways has not been comprehensively assessed. In this study, we quantified the contribution of GRKs to agonist-mediated GLP-1R internalisation and ß-arrestin recruitment profiles using cells where endogenous ß-arrestins, or non-visual GRKs were knocked out using CRISPR/Cas9 genome editing. Our results confirm the previously established atypical ß-arrestin-independent mode of GLP-1R internalisation and revealed that GLP-1R internalisation is dependent on the expression of GRKs. Interestingly, agonist-mediated GLP-1R ß-arrestin 1 and ß-arrestin 2 recruitment were differentially affected by endogenous GRK knockout with ß-arrestin 1 recruitment more sensitive to GRK knockout than ß-arrestin 2 recruitment. Moreover, individual overexpression of GRK2, GRK3, GRK5 or GRK6 in a newly generated GRK2/3/4/5/6 HEK293 cells, rescued agonist-mediated ß-arrestin 1 recruitment and internalisation profiles to similar levels, suggesting that there is no specific GRK isoform that drives these pathways. This study advances mechanistic understanding of agonist-mediated GLP-1R internalisation and provides novel insights into how GRKs may fine-tune GLP-1R signalling.

Histamine H1 Receptor-Mediated JNK Phosphorylation Is Regulated by Gq Protein-Dependent but Arrestin-Independent Pathways.

Arrestins are known to be involved not only in the desensitization and internalization of G protein-coupled receptors but also in the G protein-independent activation of mitogen-activated protein (MAP) kinases, such as extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), to regulate cell proliferation and inflammation. Our previous study revealed that the histamine H1 receptor-mediated activation of ERK is dually regulated by Gq proteins and arrestins. In this study, we investigated the roles of Gq proteins and arrestins in the H1 receptor-mediated activation of JNK in Chinese hamster ovary (CHO) cells expressing wild-type (WT) human H1 receptors, the Gq protein-biased mutant S487TR, and the arrestin-biased mutant S487A. In these mutants, the Ser487 residue in the C-terminus region of the WT was truncated (S487TR) or mutated to alanine (S487A). Histamine significantly stimulated JNK phosphorylation in CHO cells expressing WT and S487TR but not S487A. Histamine-induced JNK phosphorylation in CHO cells expressing WT and S487TR was suppressed by inhibitors against H1 receptors (ketotifen and diphenhydramine), Gq proteins (YM-254890), and protein kinase C (PKC) (GF109203X) as well as an intracellular Ca2+ chelator (BAPTA-AM) but not by inhibitors against G protein-coupled receptor kinases (GRK2/3) (cmpd101), ß-arrestin2 (ß-arrestin2 siRNA), and clathrin (hypertonic sucrose). These results suggest that the H1 receptor-mediated phosphorylation of JNK is regulated by Gq-protein/Ca2+/PKC-dependent but GRK/arrestin/clathrin-independent pathways.

The Role of G Protein-Coupled Receptors and Receptor Kinases in Pancreatic ß-Cell Function and Diabetes.

Type 2 diabetes (T2D) mellitus has emerged as a major global health concern that has accelerated in recent years due to poor diet and lifestyle. Afflicted individuals have high blood glucose levels that stem from the inability of the pancreas to make enough insulin to meet demand. Although medication can help to maintain normal blood glucose levels in individuals with chronic disease, many of these medicines are outdated, have severe side effects, and often become less efficacious over time, necessitating the need for insulin therapy. G protein-coupled receptors (GPCRs) regulate many physiologic processes, including blood glucose levels. In pancreatic ß cells, GPCRs regulate ß-cell growth, apoptosis, and insulin secretion, which are all critical in maintaining sufficient ß-cell mass and insulin output to ensure euglycemia. In recent years, new insights into the signaling of incretin receptors and other GPCRs have underscored the potential of these receptors as desirable targets in the treatment of diabetes. The signaling of these receptors is modulated by GPCR kinases (GRKs) that phosphorylate agonist-activated GPCRs, marking the receptor for arrestin binding and internalization. Interestingly, genome-wide association studies using diabetic patient cohorts link the GRKs and arrestins with T2D. Moreover, recent reports show that GRKs and arrestins expressed in the ß cell serve a critical role in the regulation of ß-cell function, including ß-cell growth and insulin secretion in both GPCR-dependent and -independent pathways. In this review, we describe recent insights into GPCR signaling and the importance of GRK function in modulating ß-cell physiology. SIGNIFICANCE STATEMENT: Pancreatic ß cells contain a diverse array of G protein-coupled receptors (GPCRs) that have been shown to improve ß-cell function and survival, yet only a handful have been successfully targeted in the treatment of diabetes. This review discusses recent advances in our understanding of ß-cell GPCR pharmacology and regulation by GPCR kinases while also highlighting the necessity of investigating islet-enriched GPCRs that have largely been unexplored to unveil novel treatment strategies.

GPCR kinases differentially modulate biased signaling downstream of CXCR3 depending on their subcellular localization.

Some G protein-coupled receptors (GPCRs) demonstrate biased signaling such that ligands of the same receptor exclusively or preferentially activate certain downstream signaling pathways over others. This phenomenon may result from ligand-specific receptor phosphorylation by GPCR kinases (GRKs). GPCR signaling can also exhibit location bias because GPCRs traffic to and signal from subcellular compartments in addition to the plasma membrane. Here, we investigated whether GRKs contributed to location bias in GPCR signaling. GRKs translocated to endosomes after stimulation of the chemokine receptor CXCR3 or other GPCRs in cultured cells. GRK2, GRK3, GRK5, and GRK6 showed distinct patterns of recruitment to the plasma membrane and to endosomes depending on the identity of the biased ligand used to activate CXCR3. Analysis of engineered forms of GRKs that localized to either the plasma membrane or endosomes demonstrated that biased CXCR3 ligands elicited different signaling profiles that depended on the subcellular location of the GRK. Each GRK exerted a distinct effect on the regulation of CXCR3 engagement of ß-arrestin, internalization, and activation of the downstream effector kinase ERK. Our work highlights a role for GRKs in location-biased GPCR signaling and demonstrates the complex interactions between ligands, GRKs, and cellular location that contribute to biased signaling.

G Protein-Coupled Receptor Kinase 2 Selectively Enhances ß-Arrestin Recruitment to the D2 Dopamine Receptor through Mechanisms That Are Independent of Receptor Phosphorylation.

The D2 dopamine receptor (D2R) 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 the phosphorylated C-terminal tail or intracellular loops. GPCR kinases (GRKs) are the primary drivers of GPCR phosphorylation, and for many receptors, receptor phosphorylation 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 the extent to which ß-arrestin recruitment to the D2R is GRK-dependent. Genetic elimination of all GRK expression decreased, but did not eliminate, agonist-stimulated ß-arrestin recruitment to the D2R or its subsequent internalization. However, these processes were rescued upon the re-introduction of various GRK isoforms in the cells with GRK2/3 also enhancing dopamine potency. Further, treatment with compound 101, a pharmacological inhibitor of GRK2/3 isoforms, decreased ß-arrestin recruitment and receptor internalization, highlighting the importance of this GRK subfamily for D2R-ß-arrestin interactions. These results were recapitulated using a phosphorylation-deficient D2R mutant, emphasizing that GRKs can enhance ß-arrestin recruitment and activation independently of receptor phosphorylation.

Atypical Chemokine Receptor 3 "Senses" CXC Chemokine Receptor 4 Activation Through GPCR Kinase Phosphorylation.

Atypical chemokine receptor 3 (ACKR3) is an arrestin-biased receptor that regulates extracellular chemokine levels through scavenging. The scavenging process restricts the availability of the chemokine agonist CXCL12 for the G protein-coupled receptor (GPCR) CXCR4 and requires phosphorylation of the ACKR3 C-terminus by GPCR kinases (GRKs). ACKR3 is phosphorylated by GRK2 and GRK5, but the mechanisms by which these kinases regulate the receptor are unresolved. Here we determined that GRK5 phosphorylation of ACKR3 results in more efficient chemokine scavenging and ß-arrestin recruitment than phosphorylation by GRK2 in HEK293 cells. However, co-activation of CXCR4-enhanced ACKR3 phosphorylation by GRK2 through the liberation of Gßγ, an accessory protein required for efficient GRK2 activity. The results suggest that ACKR3 "senses" CXCR4 activation through a GRK2-dependent crosstalk mechanism, which enables CXCR4 to influence the efficiency of CXCL12 scavenging and ß-arrestin recruitment to ACKR3. Surprisingly, we also found that despite the requirement for phosphorylation and the fact that most ligands promote ß-arrestin recruitment, ß-arrestins are dispensable for ACKR3 internalization and scavenging, suggesting a yet-to-be-determined function for these adapter proteins. Since ACKR3 is also a receptor for CXCL11 and opioid peptides, these data suggest that such crosstalk may also be operative in cells with CXCR3 and opioid receptor co-expression. Additionally, kinase-mediated receptor cross-regulation may be relevant to other atypical and G protein-coupled receptors that share common ligands. SIGNIFICANCE STATEMENT: The atypical receptor ACKR3 indirectly regulates CXCR4-mediated cell migration by scavenging their shared agonist CXCL12. Here, we show that scavenging and ß-arrestin recruitment by ACKR3 are primarily dependent on phosphorylation by GRK5. However, we also show that CXCR4 co-activation enhances the contribution of GRK2 by liberating Gßγ. This phosphorylation crosstalk may represent a common feedback mechanism between atypical and G protein-coupled receptors with shared ligands for regulating the efficiency of scavenging or other atypical receptor functions.

Novel roles for G protein-coupled receptor kinases in cardiac injury and repair.

G protein-coupled receptors (GPCRs) are key modulators of cell signaling. Multiple GPCRs are present in the heart where they regulate cardiac homeostasis including processes such as myocyte contraction, heart rate and coronary blood flow. GPCRs are pharmacological targets for several cardiovascular disorders including heart failure (HF) such as beta-adrenergic receptor (ßAR) blockers and angiotensin II receptor (AT1R) antagonists. The activity of GPCRs are finely regulated by GPCR kinases (GRKs), which phosphorylate agonist-occupied receptors and start the process of desensitization. Among the seven members of the GRK family, GRK2 and GRK5 are predominantly expressed in the heart, where they exhibit both canonical and non-canonical functions. Both kinases are known to be increased in cardiac pathologies and contribute to pathogenesis through their roles in different cellular compartments. Lowering or inhibiting their actions mediate cardioprotective effects against pathological cardiac growth and failing heart. Therefore, given their importance in cardiac dysfunction, these kinases are drawing attention as promising targets for the treatment of HF, which needs improved therapies. Over the past three decades, broad knowledge on GRK inhibition in HF has been gained by studies using genetically engineered animal models or through gene therapy with peptide inhibitors or using small molecule inhibitors. In this mini review, we summarize the work focusing on GRK2 and GRK5 but also discuss a couple of the non-abundant cardiac subtypes and their multi-functional roles in the normal and diseased heart and the potential and therapeutic targets.

Delineation of G Protein-Coupled Receptor Kinase Phosphorylation Sites within the D1 Dopamine Receptor and Their Roles in Modulating ß-Arrestin Binding and Activation.

The D1 dopamine receptor (D1R) is a G protein-coupled receptor that signals through activating adenylyl cyclase and raising intracellular cAMP levels. When activated, the D1R also recruits the scaffolding protein ß-arrestin, which promotes receptor desensitization and internalization, as well as additional downstream signaling pathways. These processes are triggered through receptor phosphorylation by G protein-coupled receptor kinases (GRKs), although the precise phosphorylation sites and their role in recruiting ß-arrestin to the D1R remains incompletely described. In this study, we have used detailed mutational and in situ phosphorylation analyses to completely identify the GRK-mediated phosphorylation sites on the D1R. Our results indicate that GRKs can phosphorylate 14 serine and threonine residues within the C-terminus and the third intracellular loop (ICL3) of the receptor, and that this occurs in a hierarchical fashion, where phosphorylation of the C-terminus precedes that of the ICL3. Using ß-arrestin recruitment assays, we identified a cluster of phosphorylation sites in the proximal region of the C-terminus that drive ß-arrestin binding to the D1R. We further provide evidence that phosphorylation sites in the ICL3 are responsible for ß-arrestin activation, leading to receptor internalization. Our results suggest that distinct D1R GRK phosphorylation sites are involved in ß-arrestin binding and activation.

Arrestin-dependent nuclear export of phosphodiesterase 4D promotes GPCR-induced nuclear cAMP signaling required for learning and memory.

G protein-coupled receptors (GPCRs) promote the expression of immediate early genes required for learning and memory. Here, we showed that ß2-adrenergic receptor (ß2AR) stimulation induced the nuclear export of phosphodiesterase 4D5 (PDE4D5), an enzyme that degrades the second messenger cAMP, to enable memory consolidation. We demonstrated that the endocytosis of ß2AR phosphorylated by GPCR kinases (GRKs) mediated arrestin3-dependent nuclear export of PDE4D5, which was critical for promoting nuclear cAMP signaling and gene expression in hippocampal neurons for memory consolidation. Inhibition of the arrestin3-PDE4D5 association prevented ß2AR-induced nuclear cAMP signaling without affecting receptor endocytosis. Direct PDE4 inhibition rescued ß2AR-induced nuclear cAMP signaling and ameliorated memory deficits in mice expressing a form of the ß2AR that could not be phosphorylated by GRKs. These data reveal how ß2AR phosphorylated by endosomal GRK promotes the nuclear export of PDE4D5, leading to nuclear cAMP signaling, changes in gene expression, and memory consolidation. This study also highlights the translocation of PDEs as a mechanism to promote cAMP signaling in specific subcellular locations downstream of GPCR activation.

A novel G protein-biased agonist at the µ opioid receptor induces substantial receptor desensitisation through G protein-coupled receptor kinase.

BACKGROUND AND PURPOSE: G protein-biased µ opioid receptor agonists have the potential to induce less receptor desensitisation and tolerance than balanced opioids. Here, we investigated if the cyclic endomorphin analogue Tyr-c[D-Lys-Phe-Tyr-Gly] (Compound 1) is a G protein-biased µ agonist and characterised its ability to induce rapid receptor desensitisation in mammalian neurones. EXPERIMENTAL APPROACH: The signalling and trafficking properties of opioids were characterised using bioluminescence resonance energy transfer assays, enzyme-linked immunosorbent assay and phosphosite-specific immunoblotting in human embryonic kidney 293 cells. Desensitisation of opioid-induced currents were studied in rat locus coeruleus neurones using whole-cell patch-clamp electrophysiology. The mechanism of Compound 1-induced µ receptor desensitisation was probed using kinase inhibitors. KEY RESULTS: Compound 1 has similar intrinsic activity for G protein signalling as morphine. As predicted for a G protein-biased µ agonist, Compound 1 induced minimal agonist-induced internalisation and phosphorylation at intracellular µ receptor serine/threonine residues known to be involved in G protein-coupled receptor kinase (GRK)-mediated desensitisation. However, Compound 1 induced robust rapid µ receptor desensitisation in locus coeruleus neurons, to a greater degree than morphine. The extent of Compound 1-induced desensitisation was unaffected by activation or inhibition of protein kinase C (PKC) but was significantly reduced by inhibition of GRK. CONCLUSION AND IMPLICATIONS: Compound 1 is a novel G protein-biased µ agonist that induces substantial rapid receptor desensitisation in mammalian neurons. Surprisingly, Compound 1-induced desensitisation was demonstrated to be GRK dependent despite its G protein bias. Our findings refute the assumption that G protein-biased agonists will evade receptor desensitisation and tolerance. LINKED ARTICLES: This article is part of a themed issue on Advances in Opioid Pharmacology at the Time of the Opioid Epidemic. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v180.7/issuetoc.

ß-arrestins and G protein-coupled receptor kinases in viral entry: A graphical review.

Viruses rely on host-cell machinery in order to invade host cells and carry out a successful infection. G-protein coupled receptor (GPCR)-mediated signaling pathways are master regulators of cellular physiological processing and are an attractive target for viruses to rewire cells during infection. In particular, the GPCR-associated scaffolding proteins ß-arrestins and GPCR signaling effectors G-protein receptor kinases (GRKs) have been identified as key cellular factors that mediate viral entry and orchestrate signaling pathways that reprogram cells for viral replication. Interestingly, a broad range of viruses have been identified to activate and/or require GPCR-mediated pathways for infection, including polyomaviruses, flaviviruses, influenza virus, and SARS-CoV-2, demonstrating that these viruses may have conserved mechanisms of host-cell invasion. Thus, GPCR-mediated pathways highlight an attractive target for the development of broad antiviral therapies.

Grk7 but not Grk1 undergoes cAMP-dependent phosphorylation in zebrafish cone photoreceptors and mediates cone photoresponse recovery to elevated cAMP.

In the vertebrate retina, phosphorylation of photoactivated visual pigments in rods and cones by G protein-coupled receptor kinases (GRKs) is essential for sustained visual function. Previous in vitro analysis demonstrated that GRK1 and GRK7 are phosphorylated by PKA, resulting in a reduced capacity to phosphorylate rhodopsin. In vivo observations revealed that GRK phosphorylation occurs in the dark and is cAMP dependent. In many vertebrates, including humans and zebrafish, GRK1 is expressed in both rods and cones while GRK7 is expressed only in cones. However, mice express only GRK1 in both rods and cones and lack GRK7. We recently generated a mutation in Grk1 that deletes the phosphorylation site, Ser21. This mutant demonstrated delayed dark adaptation in mouse rods but not in cones in vivo, suggesting GRK1 may serve a different role depending upon the photoreceptor cell type in which it is expressed. Here, zebrafish were selected to evaluate the role of cAMP-dependent GRK phosphorylation in cone photoreceptor recovery. Electroretinogram analyses of larvae treated with forskolin show that elevated intracellular cAMP significantly decreases recovery of the cone photoresponse, which is mediated by Grk7a rather than Grk1b. Using a cone-specific dominant negative PKA transgene, we show for the first time that PKA is required for Grk7a phosphorylation in vivo. Lastly, immunoblot analyses of rod grk1a-/- and cone grk1b-/- zebrafish and Nrl-/- mouse show that cone-expressed Grk1 does not undergo cAMP-dependent phosphorylation in vivo. These results provide a better understanding of the function of Grk phosphorylation relative to cone adaptation and recovery.

ß-arrestin1 and 2 exhibit distinct phosphorylation-dependent conformations when coupling to the same GPCR in living cells.

ß-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-ß-arrestin complex. However, whether ß-arrestin1 and 2 respond differently for binding to the same GPCR is still unknown. Employing GRK knockout cells and ß-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 ß-arrestin1 and 2 when bound to active PTH1R (P-R*). Moreover, we assess ß-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.

Clinical utility of lymphocyte to C-reactive protein ratio in predicting survival and postoperative complication for esophago-gastric junction cancer.

BACKGROUND: There are still no useful predictive biomarkers for esophago-gastric junction (EGJ) cancer. We compared 15 candidate inflammation-based markers and investigated the clinical impact of the selected biomarker. METHODS: One hundred three patients with EGJ cancer between 2002 and 2020 were enrolled, and associations between clinicopathological data and inflammatory biomarkers were retrospectively analyzed. Area under the curve (AUC) values of 15 candidate biomarkers were compared in receiver operating characteristic (ROC) curves regarding overall survival (OS). Clinical impacts of the selected marker were further investigated regarding long-term prognosis, postoperative complications, and preoperative chemotherapy effects. RESULTS: Lymphocyte/CRP ratio (LCR) demonstrated the highest AUC (0.68552) and was chosen as a candidate biomarker. The high LCR group (LCR >4610) demonstrated significantly better OS (p < 0.0001) and relapse-free survival (RFS) (p < 0.0001) compared with the low LCR group (LCR ≤4610), and preoperative LCR was an independent prognostic factor for both OS (HR 4.97, 95% CI:2.24-11.58; p < 0.0001) and RFS (HR 2.84, 95% CI:1.33-6.14, p = 0.007) in EGJ cancer patients. Another cut-off value was established for postoperative complications, and the incidence rates were significantly higher in the low LCR group (LCR ≤12000) than in the high LCR group (LCR >12000) for all postoperative complications, infectious complications, and surgical site infection (p = 0.013, p = 0.016, and p = 0.030, respectively). Furthermore, patients with decreased LCR after preoperative chemotherapy demonstrated significantly worse RFS compared with patients with increased LCR (p = 0.043). CONCLUSIONS: LCR is a potential biomarker to predict long-term prognosis as well as occurrence of postoperative complications in patients with EGJ cancer.

G protein-coupled receptor kinase phosphorylation of distal C-tail sites specifies ßarrestin1-mediated signaling by chemokine receptor CXCR4.

G protein-coupled receptor (GPCR) kinases (GRKs) and arrestins mediate GPCR desensitization, internalization, and signaling. The spatial pattern of GPCR phosphorylation is predicted to trigger these discrete GRK and arrestin-mediated functions. Here, we provide evidence that distal carboxyl-terminal tail (C-tail), but not proximal, phosphorylation of the chemokine receptor CXCR4 specifies ßarrestin1 (ßarr1)-dependent signaling. We demonstrate by pharmacologic inhibition of GRK2/3-mediated phosphorylation of the chemokine receptor CXCR4 coupled with site-directed mutagenesis and bioluminescence resonance energy transfer approaches that distal, not proximal, C-tail phosphorylation sites are required for recruitment of the adaptor protein STAM1 (signal-transducing adaptor molecule) to ßarr1 and focal adhesion kinase phosphorylation but not extracellular signal-regulated kinase 1/2 phosphorylation. In addition, we show that GPCRs that have similarly positioned C-tail phosphoresidues are also able to recruit STAM1 to ßarr1. However, although necessary for some GPCRs, we found that distal C-tail sites might not be sufficient to specify recruitment of STAM1 to ßarr1 for other GPCRs. In conclusion, this study provides evidence that distal C-tail phosphorylation sites specify GRK-ßarrestin-mediated signaling by CXCR4 and other GPCRs.

G protein-coupled receptor kinase 6 (GRK6) regulates insulin processing and secretion via effects on proinsulin conversion to insulin.

Recent studies identified a missense mutation in the gene coding for G protein-coupled receptor kinase 6 (GRK6) that segregates with type 2 diabetes (T2D). To better understand how GRK6 might be involved in T2D, we used pharmacological inhibition and genetic knockdown in the mouse ß-cell line, MIN6, to determine whether GRK6 regulates insulin dynamics. We show inhibition of GRK5 and GRK6 increased insulin secretion but reduced insulin processing while GRK6 knockdown revealed these same processing defects with reduced levels of cellular insulin. GRK6 knockdown cells also had attenuated insulin secretion but enhanced proinsulin secretion consistent with decreased processing. In support of these findings, we demonstrate GRK6 rescue experiments in knockdown cells restored insulin secretion after glucose treatment. The altered insulin profile appears to be caused by changes in the proprotein convertases, the enzymes responsible for proinsulin to insulin conversion, as GRK6 knockdown resulted in significantly reduced convertase expression and activity. To identify how the GRK6-P384S mutation found in T2D patients might affect insulin processing, we performed biochemical and cell biological assays to study the properties of the mutant. We found that while GRK6-P384S was more active than WT GRK6, it displayed a cytosolic distribution in cells compared to the normal plasma membrane localization of GRK6. Additionally, GRK6 overexpression in MIN6 cells enhanced proinsulin processing, while GRK6-P384S expression had little effect. Taken together, our data show that GRK6 regulates insulin processing and secretion in a glucose-dependent manner and provide a foundation for understanding the contribution of GRK6 to T2D.

G protein-coupled receptor interactions with arrestins and GPCR kinases: The unresolved issue of signal bias.

G protein-coupled receptor (GPCR) kinases (GRKs) and arrestins interact with agonist-bound GPCRs to promote receptor desensitization and downregulation. They also trigger signaling cascades distinct from those of heterotrimeric G proteins. Biased agonists for GPCRs that favor either heterotrimeric G protein or GRK/arrestin signaling are of profound pharmacological interest because they could usher in a new generation of drugs with greatly reduced side effects. One mechanism by which biased agonism might occur is by stabilizing receptor conformations that preferentially bind to GRKs and/or arrestins. In this review, we explore this idea by comparing structures of GPCRs bound to heterotrimeric G proteins with those of the same GPCRs in complex with arrestins and GRKs. The arrestin and GRK complexes all exhibit high conformational heterogeneity, which is likely a consequence of their unusual ability to adapt and bind to hundreds of different GPCRs. This dynamic behavior, along with the experimental tactics required to stabilize GPCR complexes for biophysical analysis, confounds these comparisons, but some possible molecular mechanisms of bias are beginning to emerge. We also examine if and how the recent structures advance our understanding of how arrestins parse the "phosphorylation barcodes" installed in the intracellular loops and tails of GPCRs by GRKs. In the future, structural analyses of arrestins in complex with intact receptors that have well-defined native phosphorylation barcodes, such as those installed by the two nonvisual subfamilies of GRKs, will be particularly illuminating.

GPCR kinases generate an APH1A phosphorylation barcode to regulate amyloid-ß generation.

Emerging evidence suggests that G protein-coupled receptor (GPCR) kinases (GRKs) are associated with the pathophysiology of Alzheimer's disease (AD). However, GRKs have not been directly implicated in regulation of the amyloid-ß (Aß) pathogenic cascade in AD. Here, we determine that GRKs phosphorylate a non-canonical substrate, anterior pharynx-defective 1A (APH1A), an integral component of the γ-secretase complex. Significantly, we show that GRKs generate distinct phosphorylation barcodes in intracellular loop 2 (ICL2) and the C terminus of APH1A, which differentially regulate recruitment of the scaffolding protein ß-arrestin 2 (ßarr2) to APH1A and γ-secretase-mediated Aß generation. Further molecular dynamics simulation studies reveal an interaction between the ßarr2 finger loop domain and ICL2 and ICL3 of APH1A, similar to a GPCR-ß-arrestin complex, which regulates γ-secretase activity. Collectively, these studies provide insight into the molecular and structural determinants of the APH1A-ßarr2 interaction that critically regulate Aß generation.

Nanoluciferase-based complementation assay for systematic profiling of GPCR-GRK interactions.

G protein-coupled receptor kinases (GRKs) are a family of seven soluble receptor-modifying enzymes which are essential regulators of GPCR activity. Following agonist-induced receptor activation and G protein dissociation, GRKs prime the receptor for desensitization through phosphorylation of its C terminus, which subsequently allows arrestins to bind and initiate the receptor internalization process. While GRKs constitute key GPCR-interacting proteins, to date, no method has been put forward to readily and systematically determine the preference of a specific GPCR towards the seven different GRKs (GRK1-7). This chapter describes a simple and standardized approach for systematic profiling of GRK1-7-GPCR interactions relying on the complementation of the split Nanoluciferase (NanoBiT). When applied to a set of GPCRs (MOR, 5-HT1A, B2AR, CXCR3, AVPR2, CGRPR), including two intrinsically ß-arrestin-biased receptors (ACKR2 and ACKR3), this methodology yields highly reproducible results highlighting different GRK recruitment profiles. Using this assay, further characterization of MOR, a crucial target in the development of analgesics, reveals not only its GRK fingerprint but also related kinetics and activity of various ligands for a single GRK.

Double life: How GRK2 and ß-arrestin signaling participate in diseases.

G-protein coupled receptor (GPCR) kinases (GRKs) and ß-arrestins play key roles in GPCR and non-GPCR cellular responses. In fact, GRKs and arrestins are involved in a plethora of pathways vital for physiological maintenance of inter- and intracellular communication. Here we review decades of research literature spanning from the discovery, identification of key structural elements, and findings supporting the diverse roles of these proteins in GPCR-mediated pathways. We then describe how GRK2 and ß-arrestins partake in non-GPCR signaling and briefly summarize their involvement in various pathologies. We conclude by presenting gaps in knowledge and our prospective on the promising pharmacological potential in targeting these proteins and/or downstream signaling. Future research is warranted and paramount for untangling these novel and promising roles for GRK2 and arrestins in metabolism and disease progression.