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can help to maintain normal blood glucose levels in individuals with chronic disease, many of these medicines are outdated, have severe side effects, and often become less efficacious over time, necessitating the need for insulin therapy.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.

Migrating Myeloid Cells Sense Temporal Dynamics of Chemoattractant Concentrations.

Chemoattractant-mediated recruitment of hematopoietic cells to sites of pathogen growth or tissue damage is critical to host defense and organ homeostasis. Chemotaxis is typically considered to rely on spatial sensing, with cells following concentration gradients as long as these are present. Utilizing a microfluidic approach, we found that stable gradients of intermediate chemokines (CCL19 and CXCL12) failed to promote persistent directional migration of dendritic cells or neutrophils. Instead, rising chemokine concentrations were needed, implying that temporal sensing mechanisms controlled prolonged responses to these ligands. This behavior was found to depend on G-coupled receptor kinase-mediated negative regulation of receptor signaling and contrasted with responses to an end agonist chemoattractant (C5a), for which a stable gradient led to persistent migration. These findings identify temporal sensing as a key requirement for long-range myeloid cell migration to intermediate chemokines and provide insights into the mechanisms controlling immune cell motility in complex tissue environments.

Nonenzymatic rapid control of GIRK channel function by a G protein-coupled receptor kinase.

G protein-coupled receptors (GPCRs) respond to agonists to activate downstream enzymatic pathways or to gate ion channel function. Turning off GPCR signaling is known to involve phosphorylation of the GPCR by GPCR kinases (GRKs) to initiate their internalization. The process, however, is relatively slow and cannot account for the faster desensitization responses required to regulate channel gating. Here, we show that GRKs enable rapid desensitization of the G protein-coupled potassium channel (GIRK/Kir3.x) through a mechanism independent of their kinase activity. On GPCR activation, GRKs translocate to the membrane and quench channel activation by competitively binding and titrating G protein ßγ subunits away from the channel. Of interest, the ability of GRKs to effect this rapid desensitization depends on the receptor type. The findings thus reveal a stimulus-specific, phosphorylation-independent mechanism for rapidly downregulating GPCR activity at the effector level.

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.

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.

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.

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.

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.

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.

Multisite NHERF1 phosphorylation controls GRK6A regulation of hormone-sensitive phosphate transport.

The type II sodium-dependent phosphate cotransporter (NPT2A) mediates renal phosphate uptake. The NPT2A is regulated by parathyroid hormone (PTH) and fibroblast growth factor 23, which requires Na+/H+ exchange regulatory factor-1 (NHERF1), a multidomain PDZ-containing phosphoprotein. Phosphocycling controls the association between NHERF1 and the NPT2A. Here, we characterize the critical involvement of G protein-coupled receptor kinase 6A (GRK6A) in mediating PTH-sensitive phosphate transport by targeted phosphorylation coupled with NHERF1 conformational rearrangement, which in turn allows phosphorylation at a secondary site. GRK6A, through its carboxy-terminal PDZ recognition motif, binds NHERF1 PDZ1 with greater affinity than PDZ2. However, the association between NHERF1 PDZ2 and GRK6A is necessary for PTH action. Ser162, a PKCα phosphorylation site in PDZ2, regulates the binding affinity between PDZ2 and GRK6A. Substitution of Ser162 with alanine (S162A) blocks the PTH action but does not disrupt the interaction between NHERF1 and the NPT2A. Replacement of Ser162 with aspartic acid (S162D) abrogates the interaction between NHERF1 and the NPT2A and concurrently PTH action. We used amber codon suppression to generate a phosphorylated Ser162(pSer162)-PDZ2 variant. KD values determined by fluorescence anisotropy indicate that incorporation of pSer162 increased the binding affinity to the carboxy terminus of GRK6A 2-fold compared with WT PDZ2. Molecular dynamics simulations predict formation of an electrostatic network between pSer162 and Asp183 of PDZ2 and Arg at position -1 of the GRK6A PDZ-binding motif. Our results suggest that PDZ2 plays a regulatory role in PTH-sensitive NPT2A-mediated phosphate transport and phosphorylation of Ser162 in PDZ2 modulates the interaction with GRK6A.

The evolving impact of g protein-coupled receptor kinases in cardiac health and disease.

G protein-coupled receptors (GPCRs) are important regulators of various cellular functions via activation of intracellular signaling events. Active GPCR signaling is shut down by GPCR kinases (GRKs) and subsequent ß-arrestin-mediated mechanisms including phosphorylation, internalization, and either receptor degradation or resensitization. The seven-member GRK family varies in their structural composition, cellular localization, function, and mechanism of action (see sect. II). Here, we focus our attention on GRKs in particular canonical and novel roles of the GRKs found in the cardiovascular system (see sects. III and IV). Paramount to overall cardiac function is GPCR-mediated signaling provided by the adrenergic system. Overstimulation of the adrenergic system has been highly implicated in various etiologies of cardiovascular disease including hypertension and heart failure. GRKs acting downstream of heightened adrenergic signaling appear to be key players in cardiac homeostasis and disease progression, and herein we review the current data on GRKs related to cardiac disease and discuss their potential in the development of novel therapeutic strategies in cardiac diseases including heart failure.

Receptor-proximal effectors mediating GnRH actions in the goldfish pituitary: Involvement of G protein subunits and GRKs.

In goldfish (Carassius auratus), two endogenous isoforms of gonadotropin-releasing hormone (GnRH) stimulate luteinizing hormone (LH) and growth hormone (GH) secretion. These isoforms, GnRH2 and GnRH3, act on a shared population of cell-surface GnRH receptors (GnRHRs) expressed on both gonadotrophs and somatotrophs, and can signal through unique, yet partially overlapping, suites of intracellular effectors, in a phenomenon known as functional selectivity or biased signalling. In this study, G-protein alpha (Gα) subunits were targeted with two inhibitors, YM-254890 and BIM-46187, to ascertain the contribution of specific G-protein subunits in GnRH signalling. Results with the Gαq/11-specific inhibitor YM-254890 on primary cultures of goldfish pituitary cells revealed the use of these subunits in GnRH control of both LH and GH release, as well as GnRH-induced elevations in phospho-ERK levels. Results with the pan-Gα inhibitor BIM-46187 matched those using YM-254890 in LH release but GH responses differed, indicating additional, non-Gαq/11 subunits may be involved in somatotrophs. BIM-46187 also elevated unstimulated LH and GH release suggesting that Gα subunits regulate basal hormone secretion. Furthermore, G-protein-coupled receptor kinase (GRK2/3) inhibition reduced LH responses to GnRH2 and GnRH3, and selectively enhanced GnRH2-stimulated GH release, indicating differential use of GRK2/3 in GnRH actions on gonadotrophs and somatotrophs. These findings in a primary untransformed system provide the first direct evidence to establish Gαq/11 as an obligate driver of GnRH signalling in goldfish pituitary cells, and additionally describe the differential agonist- and cell type-selective involvement of GRK2/3 in this system.

Special Issue: "G Protein-Coupled Receptor and Their Kinases in Cell Biology and Disease 2.0".

The second volume of this Special Issue, entitled "G Protein-Coupled Receptor and Their Kinases in Cell Biology and Disease 2 [...].

Suitability of GRK Antibodies for Individual Detection and Quantification of GRK Isoforms in Western Blots.

G protein-coupled receptors (GPCRs) are regulated by GPCR kinases (GRKs) which phosphorylate intracellular domains of the active receptor. This results in the recruitment of arrestins, leading to desensitization and internalization of the GPCR. Aside from acting on GPCRs, GRKs regulate a variety of membrane, cytosolic, and nuclear proteins not only via phosphorylation but also by acting as scaffolding partners. GRKs' versatility is also reflected by their diverse roles in pathological conditions such as cancer, malaria, Parkinson's-, cardiovascular-, and metabolic disease. Reliable tools to study GRKs are the key to specify their role in complex cellular signaling networks. Thus, we examined the specificity of eight commercially available antibodies targeting the four ubiquitously expressed GRKs (GRK2, GRK3, GRK5, and GRK6) in Western blot analysis. We identified one antibody that did not recognize its antigen, as well as antibodies that showed unspecific signals or cross-reactivity. Hence, we strongly recommend testing any antibody with exogenously expressed proteins to clearly confirm identity of the obtained Western blot results. Utilizing the most-suitable antibodies, we established the Western blot-based, cost-effective simple tag-guided analysis of relative protein abundance (STARPA). This method allows comparison of protein levels obtained by immunoblotting with different antibodies. Furthermore, we applied STARPA to determine GRK protein levels in nine commonly used cell lines, revealing differential isoform expression.

How Arrestins and GRKs Regulate the Function of Long Chain Fatty Acid Receptors.

FFA1 and FFA4, two G protein-coupled receptors that are activated by long chain fatty acids, play crucial roles in mediating many biological functions in the body. As a result, these fatty acid receptors have gained considerable attention due to their potential to be targeted for the treatment of type-2 diabetes. However, the relative contribution of canonical G protein-mediated signalling versus the effects of agonist-induced phosphorylation and interactions with ß-arrestins have yet to be fully defined. Recently, several reports have highlighted the ability of ß-arrestins and GRKs to interact with and modulate different functions of both FFA1 and FFA4, suggesting that it is indeed important to consider these interactions when studying the roles of FFA1 and FFA4 in both normal physiology and in different disease settings. Here, we discuss what is currently known and show the importance of understanding fully how ß-arrestins and GRKs regulate the function of long chain fatty acid receptors.

BRET-Based Biosensors to Measure Agonist Efficacies in Histamine H1 Receptor-Mediated G Protein Activation, Signaling and Interactions with GRKs and ß-Arrestins.

The histamine H1 receptor (H1R) is a G protein-coupled receptor (GPCR) and plays a key role in allergic reactions upon activation by histamine which is locally released from mast cells and basophils. Consequently, H1R is a well-established therapeutic target for antihistamines that relieve allergy symptoms. H1R signals via heterotrimeric Gq proteins and is phosphorylated by GPCR kinase (GRK) subtypes 2, 5, and 6, consequently facilitating the subsequent recruitment of ß-arrestin1 and/or 2. Stimulation of a GPCR with structurally different agonists can result in preferential engagement of one or more of these intracellular signaling molecules. To evaluate this so-called biased agonism for H1R, bioluminescence resonance energy transfer (BRET)-based biosensors were applied to measure H1R signaling through heterotrimeric Gq proteins, second messengers (inositol 1,4,5-triphosphate and Ca2+), and receptor-protein interactions (GRKs and ß-arrestins) in response to histamine, 2-phenylhistamines, and histaprodifens in a similar cellular background. Although differences in efficacy were observed for these agonists between some functional readouts as compared to reference agonist histamine, subsequent data analysis using an operational model of agonism revealed only signaling bias of the agonist Br-phHA-HA in recruiting ß-arrestin2 to H1R over Gq biosensor activation.

G protein-coupled receptor kinases are associated with Alzheimer's disease pathology.

AIM: Alzheimer's disease (AD) is characterised by extracellular deposition of amyloid-ß (Aß) in amyloid plaques and intracellular aggregation and accumulation of hyperphosphorylated tau in neurofibrillary tangles (NFTs). Although several kinases have been identified to contribute to the pathological phosphorylation of tau, kinase-targeted therapies for AD have not been successful in clinical trials. Critically, the kinases responsible for numerous identified tau phosphorylation sites remain unknown. G protein-coupled receptor (GPCR) kinases (GRKs) have recently been implicated in phosphorylation of non-GPCR substrates, for example, tubulin and α-synuclein, and in neurological disorders, including schizophrenia and Parkinson's disease. Accordingly, we investigated the involvement of GRKs in the pathophysiology of AD. METHODS: We performed a comprehensive immunohistochemical and biochemical analysis of the ubiquitously expressed GRKs, namely, GRK2, 3, 5 and 6, in postmortem human brain tissue of control subjects and AD patients. RESULTS: GRKs display unique cell-type-specific expression patterns in neurons, astrocytes and microglia. Levels of GRKs 2, 5 and 6 are specifically decreased in the CA1 region of the AD hippocampus. Biochemical evidence indicates that the GRKs differentially associate with total, soluble and insoluble pools of tau in the AD brain. Complementary immunohistochemical studies indicate that the GRKs differentially colocalise with total tau, phosphorylated tau and NFTs. Notably, GRKs 3 and 5 also colocalise with amyloid plaques. CONCLUSION: These studies establish a link between GRKs and the pathological phosphorylation and accumulation of tau and amyloid pathology in AD brains and suggest a novel role for these kinases in regulation of the pathological hallmarks of AD.

Long-term chemogenetic suppression of seizures in a multifocal rat model of temporal lobe epilepsy.

OBJECTIVE: One third of epilepsy patients do not become seizure-free using conventional medication. Therefore, there is a need for alternative treatments. Preclinical research using designer receptors exclusively activated by designer drugs (DREADDs) has demonstrated initial success in suppressing epileptic activity. Here, we evaluated whether long-term chemogenetic seizure suppression could be obtained in the intraperitoneal kainic acid rat model of temporal lobe epilepsy, when DREADDs were selectively expressed in excitatory hippocampal neurons. METHODS: Epileptic male Sprague Dawley rats received unilateral hippocampal injections of adeno-associated viral vector encoding the inhibitory DREADD hM4D(Gi), preceded by a cell-specific promotor targeting excitatory neurons. The effect of clozapine-mediated DREADD activation on dentate gyrus evoked potentials and spontaneous electrographic seizures was evaluated. Animals were systemically treated with single (.1 mg/kg/24 h) or repeated (.1 mg/kg/6 h) injections of clozapine. In addition, long-term continuous release of clozapine and olanzapine (2.8 mg/kg/7 days) using implantable minipumps was evaluated. All treatments were administered during the chronic epileptic phase and between 1.5 and 13.5 months after viral transduction. RESULTS: In the DREADD group, dentate gyrus evoked potentials were inhibited after clozapine treatment. Only in DREADD-expressing animals, clozapine reduced seizure frequency during the first 6 h postinjection. When administered repeatedly, seizures were suppressed during the entire day. Long-term treatment with clozapine and olanzapine both resulted in significant seizure-suppressing effects for multiple days. Histological analysis revealed DREADD expression in both hippocampi and some cortical regions. However, lesions were also detected at the site of vector injection. SIGNIFICANCE: This study shows that inhibition of the hippocampus using chemogenetics results in potent seizure-suppressing effects in the intraperitoneal kainic acid rat model, even 1 year after viral transduction. Despite a need for further optimization, chemogenetic neuromodulation represents a promising treatment prospect for temporal lobe epilepsy.