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.

New phosphosite-specific antibodies to unravel the role of GRK phosphorylation in dopamine D2 receptor regulation and signaling.

The dopamine D2 receptor (D2R) is the target of drugs used to treat the symptoms of Parkinson's disease and schizophrenia. The D2R is regulated through its interaction with and phosphorylation by G protein receptor kinases (GRKs) and interaction with arrestins. More recently, D2R arrestin-mediated signaling has been shown to have distinct physiological functions to those of G protein signalling. Relatively little is known regarding the patterns of D2R phosphorylation that might control these processes. We aimed to generate antibodies specific for intracellular D2R phosphorylation sites to facilitate the investigation of these mechanisms. We synthesised double phosphorylated peptides corresponding to regions within intracellular loop 3 of the hD2R and used them to raise phosphosite-specific antibodies to capture a broad screen of GRK-mediated phosphorylation. We identify an antibody specific to a GRK2/3 phosphorylation site in intracellular loop 3 of the D2R. We compared measurements of D2R phosphorylation with other measurements of D2R signalling to profile selected D2R agonists including previously described biased agonists. These studies demonstrate the utility of novel phosphosite-specific antibodies to investigate D2R regulation and signalling.

The G Protein-Coupled Receptor Kinases (GRKs) in Chemokine Receptor-Mediated Immune Cell Migration: From Molecular Cues to Physiopathology.

Although G protein-coupled receptor kinases (GRKs) have long been known to regulate G protein-coupled receptor (GPCR) desensitization, their more recently characterized functions as scaffolds and signalling adapters underscore that this small family of proteins governs a larger array of physiological functions than originally suspected. This review explores how GRKs contribute to the complex signalling networks involved in the migration of immune cells along chemokine gradients sensed by cell surface GPCRs. We outline emerging evidence indicating that the coordinated docking of several GRKs on an active chemokine receptor determines a specific receptor phosphorylation barcode that will translate into distinct signalling and migration outcomes. The guidance cues for neutrophil migration are emphasized based on several alterations affecting GRKs or GPCRs reported to be involved in pathological conditions.

Chemerin-activated functions of CMKLR1 are regulated by G protein-coupled receptor kinase 6 (GRK6) and ß-arrestin 2 in inflammatory macrophages.

Chemerin receptor (CMKLR1) is a G protein-coupled receptor (GPCR) implicated in macrophage-mediated inflammation and in several forms of human arthritis. Analogous to other GPCR, CMKLR1 is likely regulated by G protein-coupled receptor kinase (GRK) phosphorylation of intracellular domains in an activation-dependent manner, which leads to recruitment and termination of intracellular signaling via desensitization and internalization of the receptor. The ubiquitously expressed GRK family members include GRK2, GRK3, GRK5, and GRK6, but it is unknown which GRK regulates CMKLR1 cellular and signaling functions. Our data show that activation of CMKLR1 by chemerin in primary macrophages leads to signaling and functional outcomes that are regulated by GRK6 and ß-arrestin 2. We show that arrestin recruitment to CMKLR1 following chemerin stimulation is enhanced with co-expression of GRK6. Further, internalization of endogenous CMKLR1, following the addition of chemerin, is decreased in inflammatory macrophages from GRK6- and ß-arrestin 2-deficient mice. These GRK6- and ß-arrestin 2-deficient macrophages display increased migration toward chemerin and altered AKT and Extracellular-signal Related Kinase (ERK) signaling. Our findings show that chemerin-activated CMKLR1 regulation in inflammatory macrophages is largely GRK6 and ß-arrestin mediated, which may impact innate immunity and have therapeutic implications in rheumatic disease.

G Protein-Coupled Receptor Kinases in the Inflammatory Response and Signaling.

G protein-coupled receptor kinases (GRKs) are serine/threonine kinases that regulate a large and diverse class of G protein-coupled receptors (GPCRs). Through GRK phosphorylation and ß-arrestin recruitment, GPCRs are desensitized and their signal terminated. Recent work on these kinases has expanded their role from canonical GPCR regulation to include noncanonical regulation of non-GPCR and nonreceptor substrates through phosphorylation as well as via scaffolding functions. Owing to these and other regulatory roles, GRKs have been shown to play a critical role in the outcome of a variety of physiological and pathophysiological processes including chemotaxis, signaling, migration, inflammatory gene expression, etc. This diverse set of functions for these proteins makes them popular targets for therapeutics. Role for these kinases in inflammation and inflammatory disease is an evolving area of research currently pursued in many laboratories. In this review, we describe the current state of knowledge on various GRKs pertaining to their role in inflammation and inflammatory diseases.

G-protein-coupled receptor kinases in inflammation and disease.

G-protein-coupled receptor kinases (GRKs) are serine/threonine protein kinases originally discovered for their role in G-protein-coupled receptor (GPCR) phosphorylation. Recent studies have demonstrated a much broader function for this kinase family including phosphorylation of cytosolic substrates involved in cell signaling pathways stimulated by GPCRs, as well as by non-GPCRs. In addition, GRKs modulate signaling via phosphorylation-independent functions. Because of these various biochemical functions, GRKs have been shown to affect critical physiological and pathophysiological processes, and thus are considered as drug targets in diseases such as heart failure. Role of GRKs in inflammation and inflammatory diseases is an evolving area of research and several studies including work from our lab in the recent years have demonstrated critical role of GRKs in the immune system. In this review, we discuss the classical and the newly emerging functions of GRKs in the immune system and their role in inflammation and disease processes.

GRK6 phosphorylates IκBα at Ser(32)/Ser(36) and enhances TNF-α-induced inflammation.

G protein-coupled receptor kinases (GRKs) comprise a family of seven serine/threonine kinases that phosphorylate agonist-activated G protein-coupled receptors (GPCRs). It has recently been reported that GRKs regulate GPCR-independent signaling through the phosphorylation of intracellular proteins. To date, several intracellular substrates for GRK2 and GRK5 have been reported. However, those for GRK6 are poorly understood. Here we identified IκBα, a negative regulator of NF-κB signaling, as a substrate for GRK6. GRK6 directly phosphorylated IκBα at Ser(32)/Ser(36), and the kinase activity of GRK6 was required for the promotion of NF-κB signaling after TNF-α stimulation. Knockout of GRK6 in peritoneal macrophages remarkably attenuated the transcription of inflammatory genes after TNF-α stimulation. In addition, we developed a bioluminescence resonance energy transfer (BRET) probe to monitor GRK6 activity. Using this probe, we revealed that the conformational change of GRK6 was induced by TNF-α. In summary, our study demonstrates that TNF-α induces GRK6 activation, and GRK6 promotes inflammatory responses through the phosphorylation of IκBα.

A novel monoclonal antibody against human GRK6 antigen.

G protein-coupled receptor kinase 6 (GRK6) plays a universal role in receptor desensitization, by acting as a receptor-G protein interface, thereby affecting serine/threonine kinases. In this study, a 20-aa-long peptide of human GRK6 C-terminus domain was synthesized and covalently coupled to keyhole limpet hemocyanin (KLH). A mouse monoclonal antibody against human GRK6 (anti-GRK6 MAb) was successfully prepared through hybridoma technique by immunizing BALB/c mice with synthesized GRK6426-446-KLH peptides. A high specificity and affinity strain of hybridoma 5D12 were established. The titer of the purified anti-GRK6 MAb was 1.28 × 10(6) measured by indirect ELISA. Western blot and immunocytochemistry experiments were also applied to characterize the antibody specificity. Antibody absorption assays showed that the anti-GRK6 MAb can be blocked by GRK6426-446 peptides. These results indicated that the antibody could bind to GRK6 antigen specifically. This MAb provides valuable support for further studies on the functional properties of GRK6.

G Protein-coupled receptor kinase-6 interacts with activator of G protein signaling-3 to regulate CXCR2-mediated cellular functions.

The IL-8 (CXCL8) receptors CXCR1 and CXCR2 couple to Gαi to induce leukocyte recruitment and activation at sites of inflammation. We recently showed that CXCR1 couples predominantly to the G protein-coupled receptor kinase (GRK)2, whereas CXCR2 interacts with GRK6 to regulate cellular responses. In addition to G protein-coupled receptors, GRKs displayed a more diverse protein/protein interaction in cells. In this study, we sought to identify GRK6 binding partner(s) that may influence CXCL8 activities, using RBL-2H3 cells stably expressing CXCR1 (RBL-CXCR1) or CXCR2 (RBL-CXCR2), as well as human and murine neutrophils. Our data demonstrated that, upon CXCR2 activation, GRK6 interacts with activator of G protein signaling (AGS)3 and Gαi2 to form a GRK6/AGS3/Gαi2 complex. This complex is time dependent and peaked at 2-3 min postactivation. GTPγS pretreatment blocked GRK6/AGS3/Gαi2 formation, suggesting that this assembly depends on G protein activation. Surprisingly, CXCR2 activation induced AGS3 phosphorylation in a PKC-dependent, but GRK6-independent, fashion. Overexpression of AGS3 in RBL-CXCR2 significantly inhibited CXCL8-induced Ca(2+) mobilization, phosphoinositide hydrolysis, and chemotaxis. In contrast, short hairpin RNA inhibition of AGS3 enhanced CXCL8-induced Ca(2+) mobilization, receptor resistance to desensitization, and recycling to the cell surface, with no effect on receptor internalization. Interestingly, RBL-CXCR2-AGS3(-/-) cells displayed a significant increase in CXCR2 expression on the cell surface but decreased ERK1/2 and P38 MAPK activation. Taken together, these results indicate that GRK6 complexes with AGS3-Gαi2 to regulate CXCR2-mediated leukocyte functions at different levels, including downstream effector activation, receptor trafficking, and expression at the cell membrane.