G-protein Receptor Kinase Research Articles

G protein–receptor kinases 5/6 are the key regulators of G protein–coupled receptor 35–arrestin interactions

Human G protein-coupled receptor 35 is regulated by agonist-mediated phosphorylation of a set of five phospho-acceptor amino acids within its C-terminal tail. Alteration of both Ser300 and Ser303 to alanine in the GPR35a isoform greatly reduces the ability of receptor agonists to promote interactions with arrestin adapter proteins. Here, we have integrated the use of cell lines genome edited to lack expression of combinations of G protein receptor kinases (GRKs), selective small molecule inhibitors of subsets of these kinases, and antisera able to specifically identify either human GPR35a or mouse GPR35 only when Ser300 and Ser303 (orce; the equivalent residues in mouse GPR35) have become phosphorylated to demonstrate that GRK5 and GRK6 cause agonist-dependent phosphorylation of these residues. Extensions of these studies demonstrated the importance of the GRK5/6-mediated phosphorylation of these amino acids for agonist-induced internalization of the receptor. Homology and predictive modeling of the interaction of human GPR35 with GRKs showed that the N terminus of GRK5 is likely to dock in the same methionine pocket on the intracellular face of GPR35 as the C terminus of the α5 helix of Gα13 and, that while this is also the case for GRK6, GRK2 and GRK3 are unable to do so effectively. These studies provide unique and wide-ranging insights into modes of regulation of GPR35, a receptor that is currently attracting considerable interest as a novel therapeutic target in diseases including ulcerative colitis.

Endothelial APC/PAR1 distinctly regulates cytokine-induced pro-inflammatory VCAM-1 expression.

Introduction: Dysfunction of the endothelium impairs its' protective role and promotes inflammation and progression of vascular diseases. Activated Protein C (APC) elicits endothelial cytoprotective responses including barrier stabilization, anti-inflammatory and anti-apoptotic responses through the activation of the G protein-coupled receptor (GPCR) protease-activated receptor-1 (PAR1) and is a promising therapeutic. Despite recent advancements in developing new Activated protein C variants with clinical potential, the mechanism by which APC/PAR1 promotes different cytoprotective responses remains unclear and is important to understand to advance Activated protein C and new targets as future therapeutics. Here we examined the mechanisms by which APC/PAR1 attenuates cytokine-induced pro-inflammatory vascular cell adhesion molecule (VCAM-1) expression, a key mediator of endothelial inflammatory responses. Methods: Quantitative multiplexed mass spectrometry analysis of Activated protein C treated endothelial cells, endothelial cell transcriptomics database (EndoDB) online repository queries, biochemical measurements of protein expression, quantitative real-time polymerase chain reaction (RT-qPCR) measurement of mRNA transcript abundance, pharmacological inhibitors and siRNA transfections of human cultured endothelial cells. Results: Here we report that Activated Protein C modulates phosphorylation of tumor necrosis factor (TNF)-α signaling pathway components and attenuates of TNF-α induced VCAM-1 expression independent of mRNA stability. Unexpectedly, we found a critical role for the G protein-coupled receptor co-receptor sphingosine-1 phosphate receptor-1 (S1PR1) and the G protein receptor kinase-2 (GRK2) in mediating APC/PAR1 anti-inflammatory responses in endothelial cells. Discussion: This study provides new knowledge of the mechanisms by which different APC/PAR1 cytoprotective responses are mediated through discrete β-arrestin-2-driven signaling pathways modulated by specific G protein-coupled receptor co-receptors and GRKs.

Characterizing Adrenergic Regulation of Glucose Transporter 4-Mediated Glucose Uptake and Metabolism in the Heart

Whereas adrenergic stimulation promotes cardiac function that demands more fuel and energy, how this receptor controls cardiac glucose metabolism is not defined. This study shows that the cardiac β2 adrenoreceptor (β2AR) is required to increase glucose transporter 4 (GLUT4)-mediated glucose uptake in myocytes and glucose oxidation in working hearts via activating the cardiac β2AR and promotes the G inhibitory-phosphoinositide 3-kinase-protein kinase B cascade to increase phosphorylation of TBC1D4 (aka AS160), a Rab guanosine triphosphatase-activating protein, which is a key enzyme to mobilize GLUT4. Furthermore, deleting G-protein receptor kinase phosphorylation sites of β2AR blocked adrenergic stimulation of GLUT4-mediated glucose uptake in myocytes and hearts. This study defines a molecular pathway that controls cardiac GLUT4-mediated glucose uptake and metabolism under adrenergic stimulation.

Raf kinase inhibitory protein reduces bradykinin receptor desensitization.

Inflammatory hyperalgesia represents a nociceptive phenotype that can become persistent in nature through dynamic protein modifications. However, a large gap in knowledge exists concerning how the integration of intracellular signaling molecules coordinates a persistent inflammatory phenotype. Herein, we demonstrate that Raf Kinase Anchoring Protein (RKIP) interrupts a vital canonical desensitization pathway to maintain bradykinin (BK) receptor activation in primary afferent neurons. Biochemical analyses of primary neuronal cultures indicate bradykinin‐stimulated PKC phosphorylation of RKIP at Ser153. Furthermore, BK exposure increases G‐protein Receptor Kinase 2 (GRK2) binding to RKIP, inhibiting pharmacological desensitization of the BK receptor. Additional studies found that molecular RKIP down‐regulation increases BK receptor desensitization in real‐time imaging of primary afferent neurons, identifying a key pathway integrator in the desensitization process that controls multiple GRK2‐sensitive G‐protein coupled receptors. Therefore, RKIP serves as an integral scaffolding protein that inhibits BK receptor desensitization.

G protein-coupled receptor kinase 3 modulates mesenchymal stem cell proliferation and differentiation through sphingosine-1-phosphate receptor regulation

BackgroundThe bone marrow niche supports hematopoietic cell development through intimate contact with multipotent stromal mesenchymal stem cells; however, the intracellular signaling, function, and regulation of such supportive niche cells are still being defined. Our study was designed to understand how G protein receptor kinase 3 (GRK3) affects bone marrow mesenchymal stem cell function by examining primary cells from GRK3-deficient mice, which we have previously published to have a hypercellular bone marrow and leukocytosis through negative regulation of CXCL12/CXCR4 signaling.MethodsMurine GRK3-deficient bone marrow mesenchymal stromal cells were harvested and cultured to differentiate into three lineages (adipocyte, chondrocyte, and osteoblast) to confirm multipotency and compared to wild type cells. Immunoblotting, modified-TANGO experiments, and flow cytometry were used to further examine the effects of GRK3 deficiency on bone marrow mesenchymal stromal cell receptor signaling. Microcomputed tomography was used to determine trabecular and cortical bone composition of GRK3-deficient mice and standard ELISA to quantitate CXCL12 production from cellular cultures.ResultsGRK3-deficient, bone marrow-derived mesenchymal stem cells exhibit enhanced and earlier osteogenic differentiation in vitro. The addition of a sphingosine kinase inhibitor abrogated the osteogenic proliferation and differentiation, suggesting that sphingosine-1-phosphate receptor signaling was a putative G protein-coupled receptor regulated by GRK3. Immunoblotting showed prolonged ERK1/2 signaling after stimulation with sphingosine-1-phosphate in GRK3-deficient cells, and modified-TANGO assays suggested the involvement of β-arrestin-2 in sphingosine-1-phosphate receptor internalization.ConclusionsOur work suggests that GRK3 regulates sphingosine-1-phosphate receptor signaling on bone marrow mesenchymal stem cells by recruiting β-arrestin to the occupied GPCR to promote internalization, and lack of such regulation affects mesenchymal stem cell functionality.

Farnesylation and lipid unsaturation are critical for the membrane binding of the C-terminal segment of G-Protein Receptor Kinase 1

Many proteins are modified by the covalent addition of different types of lipids, such as myristoylation, palmitoylation and prenylation. Lipidation is expected to promote membrane association of proteins. Visual phototransduction involves many lipid-modified proteins. The G-Protein-coupled receptor of rod photoreceptors, rhodopsin, is inactivated by G-Protein-coupled Receptor Kinase 1 (GRK1). The C-terminus of GRK1 is farnesylated and its truncation has been shown to result in a very high decrease of its enzymatic activity, most likely because of the loss of its membrane localization. Little information is available on the membrane binding of GRK1 as well as of most prenylated proteins. Measurements of the membrane binding of the non-farnesylated and farnesylated C-terminal segment of GRK1 were thus performed using lipids typical of those found in rod outer segment disk membranes. Their random coil secondary structure was determined using circular dichroism and infrared spectroscopy. The non-farnesylated C-terminal segment of GRK1 has no surface activity. In contrast, the farnesylated C-terminal segment of GRK1 shows a particularly strong binding to lipid monolayers bearing at least one unsaturated fatty acyl chain. No binding is observed in the presence of monolayers of saturated phospholipids, in agreement with the low affinity of farnesylated Ras proteins for lipids in the liquid-ordered state. Altogether, these data demonstrate that the farnesyl group of the C-terminal segment of GRK1 is mandatory for its membrane binding, which is favored by particular lipids or lipid mixtures. This information will also be useful for the understanding of the membrane binding of other prenylated proteins.

Light-Mediated Inhibition of Colonic Smooth Muscle Constriction and Colonic Motility via Opsin 3.

Opsin photoreceptors outside of the central nervous system have been shown to mediate smooth muscle photorelaxation in several organs. We hypothesized that opsin receptor activation in the colon would have a similar effect and influence colonic motility. We detected Opsin 3 (OPN3) protein expression in the colonic wall and demonstrated that OPN3 was present in enteric neurons in the muscularis propria of the murine colon. Precontracted murine colon segments demonstrated blue light (BL) -mediated relaxation ex vivo. This photorelaxation was wavelength specific and was increased with the administration of the chromophore 9-cis retinal and a G protein receptor kinase 2 (GRK2) inhibitor. Light-mediated relaxation of the colon was not inhibited by L-NAME or tetrodotoxin (TTX). Furthermore, BL exposure in the presence of 9-cis retinal decreased the frequency of colonic migrating motor complexes (CMMC) in spontaneously contracting mouse colons ex vivo. These results demonstrate for the first time a receptor-mediated photorelaxation of colonic smooth muscle and implicate opsins as possible new targets in the treatment of spasmodic gastrointestinal dysmotility.

Constitutive activation of S1P receptors at the trans-Golgi network is required for surface transport carrier formation

SummaryThe importance of the G-protein βγ subunits in the regulation of cargo transport from the trans-Golgi network (TGN) to the plasma membrane (PM) is well accepted; however, the molecular mechanism underlying the G-protein activation at the TGN remains unclear. We show here that sphingosine 1-phosphate (S1P) receptors at the PM were trafficked to the TGN in response to a surface transport cargo, temperature-sensitive vesicular stomatitis virus glycoprotein tagged with green fluorescent protein accumulation in the Golgi. The receptor internalization occurred in an S1P-independent manner but required phosphorylation by G-protein receptor kinase 2 and β-arrestin association before internalization. Continuously activated S1P receptors in a manner dependent on S1P at the TGN kept transmitting G-protein signals including the βγ subunits supply necessary for transport carrier formation at the TGN destined for the PM.

Cell therapy rescues aging-induced beta-1 adrenergic receptor and GRK2 dysfunction in the coronary microcirculation.

Our past study showed that coronary arterioles isolated from adipose-derived stromal vascular fraction (SVF)-treated rats showed amelioration of the age-related decrease in vasodilation to beta-adrenergic receptor (β-AR) agonist and improved β-AR-dependent coronary flow and microvascular function in a model of advanced age. We hypothesized that intravenously (i.v.) injected SVF improves coronary microvascular function in aged rats by re-establishing the equilibrium of the negative regulators of the internal adrenergic signaling cascade, G-protein receptor kinase 2 (GRK2) and G-alpha inhibitory (Gαi) proteins, back to youthful levels. Female Fischer-344 rats aged young (3 months, n = 24), old (24 months, n = 26), and old animals that received 1 × 107 green fluorescent protein (GFP+) SVF cells (O + SVF, n = 11) 4 weeks prior to sacrifice were utilized. Overnight urine was collected prior to sacrifice for catecholamine measurements. Cardiac samples were used for western blotting while coronary arterioles were isolated for pressure myography studies, immunofluorescence staining, and RNA sequencing. Coronary microvascular levels of the β1 adrenergic receptor are decreased with advancing age, but this decreased expression was rescued by SVF treatment. Aging led to a decrease in phosphorylated GRK2 in cardiomyocytes vs. young control with restoration of phosphorylation status by SVF. In vessels, there was no change in genetic transcription (RNAseq) or protein expression (immunofluorescence); however, inhibition of GRK2 (paroxetine) led to improved vasodilation to norepinephrine in the old control (OC) and O + SVF, indicating greater GRK2 functional inhibition of β1-AR in aging. SVF works to improve adrenergic-mediated vasodilation by restoring the β1-AR population and mitigating signal cascade inhibitors to improve vasodilation.

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.

Articles of Significant Interest in This Issue

JC polyomavirus (JCPyV) usurps serotonin 5-hydroxytryptamine (5-HT) subtype 2 (2A, 2B, and 2C) family receptors (5-HT2Rs) for entry by clathrin-mediated endocytosis. Internalization of JCPyV requires the 5-HT2R scaffolding protein β-arrestin, yet the process by which β-arrestin is recruited to 5-HT2Rs to facilitate entry remained unclear. Mayberry et al. (e02139-20) found that G protein receptor kinase 2 (GRK2) helps facilitate interactions between β-arrestin and specific β-arrestin binding motifs within 5-HT2Rs for JCPyV entry. This work further characterizes the cellular factors that promote JCPyV internalization and the resultant infection, providing improved insights into the mechanisms of infection by nonenveloped viruses.

GRKs as Key Modulators of Opioid Receptor Function.

Understanding the link between agonist-induced phosphorylation of the mu-opioid receptor (MOR) and the associated physiological effects is critical for the development of novel analgesic drugs and is particularly important for understanding the mechanisms responsible for opioid-induced tolerance and addiction. The family of G protein receptor kinases (GRKs) play a pivotal role in such processes, mediating phosphorylation of residues at the C-tail of opioid receptors. Numerous strategies, such as phosphosite specific antibodies and mass spectrometry have allowed the detection of phosphorylated residues and the use of mutant knock-in mice have shed light on the role of GRK regulation in opioid receptor physiology. Here we review our current understanding on the role of GRKs in the actions of opioid receptors, with a particular focus on the MOR, the target of most commonly used opioid analgesics such as morphine or fentanyl.

Regulation of Kappa Opioid Receptor Inactivation Depends on Sex and Cellular Site of Antagonist Action.

The prototypical member of the receptor-inactivating kappa opioid receptor (KOR) antagonists, norbinaltorphimine (norBNI), produces prolonged receptor inactivation by a cJun kinase mechanism. These antagonists have potential therapeutic utility in the treatment of stress disorders; however, additional preclinical characterization is necessary to understand important aspects of their action. In this study, we report that norBNI does not work as effectively in female mice as in males because of estrogen regulation of G protein receptor kinase (GRK); pretreatment of ovary-intact female mice with the selective GRK2/3 inhibitor, Compound 101, made females equally sensitive to norBNI as males. Prior observations suggested that in vivo treatment with norBNI does not produce long-lasting inhibition of KOR regulation of dopamine release in the nucleus accumbens. We assessed the persistence of norBNI receptor inactivation in subcellular compartments. Fast-scan cyclic voltammetry recordings confirmed that presynaptic inhibition of dopamine release by the KOR agonist U69,593 was not blocked by in vivo pretreatment with norBNI under conditions that prevented KOR-mediated aversion and analgesia. We employed a novel in vivo proxy sensor of KOR activation, adenovirus associated double floxed inverted-HyPerRed, and demonstrated that KOR activation stimulates cJun kinase-dependent reactive oxygen species (ROS) production in somatic regions of ventral tegmental area dopamine neurons, but did not activate ROS production in dopamine terminals. The compartment selective action helps explain how dopamine somatic, but not terminally expressed, KORs are inactivated by norBNI. These results further elucidate molecular signaling mechanisms mediating receptor-inactivating KOR antagonist action and advance medication development for this novel class of stress-resilience medications. SIGNIFICANCE STATEMENT: Kappa opioid receptor (KOR) antagonists are being developed as novel proresilience therapeutics for the treatment of mood and substance use disorders. This study showed that the long-acting KOR antagonists are affected by both the sex of the animal and the subcellular compartment in which the receptor is expressed.

Agonist-induced formation of unproductive receptor-G12 complexes

G proteins are activated when they associate with G protein-coupled receptors (GPCRs), often in response to agonist-mediated receptor activation. It is generally thought that agonist-induced receptor-G protein association necessarily promotes G protein activation and, conversely, that activated GPCRs do not interact with G proteins that they do not activate. Here we show that GPCRs can form agonist-dependent complexes with G proteins that they do not activate. Using cell-based bioluminescence resonance energy transfer (BRET) and luminescence assays we find that vasopressin V2 receptors (V2R) associate with both Gs and G12 heterotrimers when stimulated with the agonist arginine vasopressin (AVP). However, unlike V2R-Gs complexes, V2R-G12 complexes are not destabilized by guanine nucleotides and do not promote G12 activation. Activating V2R does not lead to signaling responses downstream of G12 activation, but instead inhibits basal G12-mediated signaling, presumably by sequestering G12 heterotrimers. Overexpressing G12 inhibits G protein receptor kinase (GRK) and arrestin recruitment to V2R and receptor internalization. Formyl peptide (FPR1 and FPR2) and Smoothened (Smo) receptors also form complexes with G12 that are insensitive to nucleotides, suggesting that unproductive GPCR-G12 complexes are not unique to V2R. These results indicate that agonist-dependent receptor-G protein association does not always lead to G protein activation and may in fact inhibit G protein activation.

Chronic Treatment with Morphine Disrupts Acute Kinase-Dependent Desensitization of GPCRs

Based on studies using mutations of the µ-opioid receptor (MOR), phosphorylation of multiple sites on the C-terminus has been recognized as a critical step underlying acute desensitization and the development of cellular tolerance. The aim of this study is to explore which kinases mediate desensitization of MOR in brain slices from drug-naïve and morphine-treated animals. Whole-cell recordings from locus coeruleus neurons were made, and the agonist-induced increase in potassium conductance was measured. In slices from naïve animals, pharmacological inhibition of G-protein receptor kinase (GRK2/3) with compound 101 blocked acute desensitization. Following chronic treatment with morphine, compound 101 was less effective at blocking acute desensitization. Compound 101 blocked receptor internalization in tissue from both naïve and morphine-treated animals, suggesting that GRK2/3 remained active. Kinase inhibitors aimed at blocking protein kinase C and c-Jun N-terminal kinase had no effect on desensitization in tissue taken from naïve animals. However, in slices taken from morphine-treated animals, the combination of these blockers along with compound 101 was required to block acute desensitization. Acute desensitization of the potassium conductance induced by the somatostatin receptor was also blocked by compound 101 in slices from naïve but not morphine-treated animals. As was observed with MOR, it was necessary to use the combination of kinase inhibitors to block desensitization of the somatostatin receptor in slices from morphine-treated animals. The results show that chronic treatment with morphine results in a surprising and heterologous adaptation in kinase-dependent desensitization. SIGNIFICANCE STATEMENT: The results show that chronic treatment with morphine induced heterologous adaptations in kinase regulation of G protein coupled receptor (GPCR) desensitization. Although the canonical mechanism for acute desensitization through phosphorylation by G protein-coupled receptor kinase is supported in tissue taken from naïve animals, following chronic treatment with morphine, the acute kinase-dependent desensitization of GPCRs is disrupted such that additional kinases, including protein kinase C and c-Jun N-terminal kinase, contribute to desensitization.

Characterization of GRK5 as a novel regulator of rhabdomyosarcoma tumor cell growth and self-renewal.

Rhabdomyosarcoma (RMS) is the most common soft-tissue pediatric sarcoma. Clinical outcomes for RMS patients with relapsed or metastatic disease remain poor. Treatment options remain limited, presenting an urgent need for novel therapeutic targets. Using a high-throughput siRNA screen against the human kinome, we identified GRK5, a G-protein receptor kinase, as a novel regulator of RMS tumor cell growth and self-renewal. Through functional assays in vitro and in vivo, we show that GRK5 regulates cell cycle in a kinase-independent manner to promote RMS tumor cell growth. NFAT1 expression is regulated by GRK5 in a kinase independent manner, and loss of NFAT1 phenocopies GRK5 loss-of-function effects on the cell cycle alterations. Self-renewal of tumor propagating cells (TPCs) is thought to give rise to tumor relapse. We show that loss of GRK5 results in a significant reduction of RMS self-renewal capacity in part due to increased cell death. Treatment of human RMS xenografts in mice with CCG-215022, a GRK5-selective inhibitor, results in reduced tumor growth and self-renewal in both major subtypes of RMS. GRK5 represents a novel therapeutic target for the treatment of RMS.

GRK Regulation of Beta2 Adrenergic Receptor Mediated Gene Transcription: Impacts on Learning and Memory

BackgroundAdrenergic signaling plays critical roles in health and disease both peripherally and centrally. In the central nervous system, beta adrenergic receptors are broadly expressed and respond to norepinephrine (NE) signaling from neurons projected from the Locus Coeruleous (LC). NE is known to regulate attention, arousal, and acuity as well as fear learning in health and is implicated in neurodegenerative disease and post‐traumatic stress disorder (PTSD). Beta2 adrenergic receptors (B2ARs) are known to be involved in long term potentiation through Gs mediated activation of protein kinase A (PKA) which regulates calcium channels or through forming complexes with AMPA receptors. Recently published work from our lab has shown that functionally distinct and selectively phosphorylated subpopulations of B2ARs exist in a single neuron. These subpopulations of B2ARs are exclusively phosphorylated at either a PKA (S261/262) or g‐protein receptor kinase (GRK) (S355/356) site on the c‐terminus of the receptor and display distinct agonist‐induced receptor intracellular trafficking. Here we investigate the distinct signals associated with the selective phosphorylation of B2ARs at the GRK and PKA site and seek to elucidate the functional implications of the signals in vitro and in vivo.HypothesisWe hypothesize that stimulation of B2AR causes phosphorylation of the receptor at either PKA or GRK phosphorylation sites. Phosphorylation of the GRK site leads to receptor internalization and delivers signaling to the nucleus, which can regulate gene transcription while phosphorylation of the PKA site does not.MethodsOverexpression of wild type B2AR or receptors with serine to alanine mutations of the GRK (S355/356A) or PKA (S261/262A) phosphorylation sites in neurons as well as pharmacologic inhibitors of PKA or GRK were used to study receptor trafficking and signaling. Förster resonance energy transfer (FRET) biosensors ICUE3 expressed at the plasma membrane or the nucleus were used to detect cAMP signaling. Knockin mice expressing mutant receptor were used to examine learning and memory in the Morris water maze paradigm.ResultsStimulation of B2AR leads to distinct membrane trafficking based on phosphorylation status of the receptor. Phosphorylation of the GRK but not PKA site on B2AR is necessary for internalization of the receptor and this internalized receptor is in an active conformation. GRK phosphorylation of B2AR is also necessary for increased cAMP levels in the nucleus and transcription of cFOS, FOSB, RHOB, and OLIG2, genes associated with learning and memory. Knockin mice expressing B2AR with a S355/356A mutation showed decreased spatial learning and memory.Conclusion and SignificanceThese data demonstrate that GRK‐dependent B2AR signaling can regulate gene transcription and that loss of this internal signaling affects learning and memory in mice. These data offer new mechanistic insight into B2AR signaling in the brain that could have implications for both our understanding of learning and memory and for neurological diseases and PTSD.Support or Funding InformationThis work was funded by NIH T32GM099608 and GM GM129376

Targeting GPCR GRK2 signaling as a novel modulator of osteoarthritis

Purpose: Osteoarthritis (OA) is a prevalent and chronic degenerative joint disease which causes pain and disability, with no disease-modifying treatment. Cartilage degeneration and bone remodeling are key characteristics of OA. The pathogenesis of OA is a complex mechanism. G protein-coupled receptors (GPCR) are important regulators of chondrocytes and osteoblasts. While G protein receptor kinase-2 (GRK2) signaling plays an important pathological role in many diseases, its role in cartilage degeneration and subchondral bone sclerosis in OA remains unknown.

G-protein receptor kinase 2 inhibition attenuates osteoarthritis progression and promotes parathyroid hormone chondroprotection

Purpose: This study aimed to evaluate the role of GRK2 inhibition in PTH1R resensitization and PTH-mediated chondroprotection in OA. Osteoarthritis (OA) is a progressive degenerative joint disease, highlighted by chondrocyte hypertrophy, cartilage degeneration, subchondral bone remodeling, and synovial inflammation. Current treatments are limited to palliative pain management therapies with little potential to prevent further disease progression. Parathyroid hormone (PTH) has demonstrated chondroprotective and chondroregenerative effects in OA mice.

Melanopsin Carboxy-terminus phosphorylation plasticity and bulk negative charge, not strict site specificity, achieves phototransduction deactivation.

Melanopsin is a visual pigment expressed in a small subset of ganglion cells in the mammalian retina known as intrinsically photosensitive retinal ganglion cells (ipRGCs) and is implicated in regulating non-image forming functions such as circadian photoentrainment and pupil constriction and contrast sensitivity in image formation. Mouse melanopsin's Carboxy-terminus (C-terminus) possesses 38 serine and threonine residues, which can potentially serve as phosphorylation sites for a G-protein Receptor Kinase (GRK) and be involved in the deactivation of signal transduction. Previous studies suggest that S388, T389, S391, S392, S394, S395 on the proximal region of the C-terminus of mouse melanopsin are necessary for melanopsin deactivation. We expressed a series of mouse melanopsin C-terminal mutants in HEK293 cells and using calcium imaging, and we found that the necessary cluster of six serine and threonine residues, while being critical, are insufficient for proper melanopsin deactivation. Interestingly, the additional six serine and threonine residues adjacent to the required six sites, in either proximal or distal direction, are capable of restoring wild-type deactivation of melanopsin. These findings suggest an element of plasticity in the molecular basis of melanopsin phosphorylation and deactivation. In addition, C-terminal chimeric mutants and molecular modeling studies support the idea that the initial steps of deactivation and β-arrestin binding are centered around these critical phosphorylation sites (S388-S395). The degree of functional versatility described in this study, along with ipRGC biophysical heterogeneity and the possible use of multiple signal transduction cascades, might contribute to the diverse ipRGC light responses for use in non-image and image forming behaviors, even though all six sub types of ipRGCs express the same melanopsin gene OPN4.