Quantitation of Plasma Membrane (G Protein-Coupled) Receptor Trafficking in Cultured Cells.

Measuring the functional behavior of G protein-coupled receptors (GPCRs) has been a major focus of academic and pharmaceutical research for many decades. These efforts have led to the development of many assays to measure the downstream effects of ligand binding on receptor activity. In this chapter, we describe an internalization/recycling assay that can be used to track changes in receptor number at the plasma membrane. Used in concert with other assays, this antibody-based technique can provide dynamic information on GPCR activation by receptor-specific ligands.

G protein coupled receptor kinase 5 modifies the nucleolar stress response activated by actinomycin D.

G protein coupled receptor kinase 5 (GRK5) is localized within the nucleus and moderates functions such as DNA transcription, in addition to its localization at the plasma membrane. In this report, we show that GRK5 modifies the nucleolar stress response activated by the DNA polymerase inhibitor, actinomycin D (ActD). We show an increased sensitivity to the apoptotic effects of ActD on cervical HeLa cells and the breast cancer cell line MDA MB 231 with reduced protein expression of GRK5. We also tested two types of breast cancer cells (MDA MB 231 and MCF7 cells) and found that the rate of response to ActD varied between them because they have innate differences in the protein expression of GRK5. We also found that GRK5 phosphorylates nucleophosmin (NPM1) at T199 before and during the early stages of ActD treatment. Phosphorylation at T199 increases the ability of NPM1 to interact with p14ARF in vitro, which may affect the protein expression levels of p14ARF. We found that the expression levels of p14ARF were lower in the cells transfected with the control shRNA, but higher in cells transfected with GRK5 shRNA. Collectively, this suggests that GRK5 modifies the nucleolar stress response associated with ActD.

G protein-coupled receptor kinase 2 modifies the ability of Caenorhabditis elegans to survive oxidative stress.

Survival and adaptation to oxidative stress is important for many organisms, and these occur through the activation of many different signaling pathways. In this report, we showed that Caenorhabditis (C.) elegans G protein-coupled receptor kinases modified the ability of the organism to resist oxidative stress. In acute oxidative stress studies using juglone, loss-of-function grk-2 mutants were more resistant to oxidative stress compared with loss-of-function grk-1 mutants and the wild-type N2 animals. This effect was Ce-AKT-1 dependent, suggesting that Ce-GRK2 adjusted C. elegans oxidative stress resistance through the IGF/insulin-like signaling (IIS) pathway. Treating C. elegans with a GRK2 inhibitor, the selective serotonin reuptake inhibitor paroxetine, resulted in increased acute oxidative stress resistance compared with another selective serotonin reuptake inhibitor, fluoxetine. In chronic oxidative stress studies with paraquat, both grk-1 and grk-2 mutants had longer lifespan compared with the wild-type N2 animals in stress. In summary, this research showed the importance of both GRKs, especially GRK2, in modifying oxidative stress resistance.

IDPs and their complexes in GPCR and nuclear receptor signaling.

G protein-coupled receptors (GPCRs) and Nuclear Receptors (NRs) are two signaling machineries that are involved in major physiological processes and, as a consequence, in a substantial number of diseases. Therefore, they actually represent two major targets for drugs with potential applications in almost all public health issues. Full exploitation of these targets for therapeutic purposes nevertheless requires opening original avenues in drug design, and this in turn implies a better understanding of the molecular mechanisms underlying their functioning. However, full comprehension of how these complex systems function and how they are deregulated in a physiopathological context is obscured by the fact that these proteins include a substantial number of disordered regions that are central to their mechanism of action but whose structural and functional properties are still largely unexplored. In this chapter, we describe how these intrinsically disordered regions (IDR) or proteins (IDP) intervene, control and finely modulate the thermodynamics of complexes involved in GPCR and NR regulation, which in turn triggers a multitude of cascade of events that are exquisitely orchestrated to ultimately control the biological output.

G protein coupled receptor kinases modulate Caenorhabditis elegans reactions to heat stresses.

In this report, we explored if G protein coupled receptor kinases (GRKs) can help modulate the heat stress responses of Caenorhabditis (C.) elegans. Loss of function grk-2 C. elegans mutants were more tolerant to increases in heat and display an ability for heat stress-associated hormesis at a longer exposure time unlike the wild type N2 animals and the loss of function grk-1 C. elegans mutants. The loss of function grk-1 mutants recovered more from acute heat stress compared to the wild type N2 animals. Animals with low Ce-GRK2 protein expression showed increased DAF-16 nuclear localization during the early stages of heat stress exposure compared to the other RNAi-treated animals, demonstrating altered insulin/insulin-like growth factor signaling (IIS) pathway activity in response to the stress. pdk-1 and akt-1 may play key roles in conjunction with Ce-GRK2 in the heat stress response. Collectively, these findings demonstrate that GRKs influence C. elegans heat stress behaviors.

[Bone-edge electroacupuncture relieves pain by regulating expression of GRK5, ß-arrestin 2 and PKCα proteins in locus coeruleus in bone cancer pain rats with morphine tolerance].

OBJECTIVE: To observe the effect of bone-edge electroacupuncture (EA) intervention on mechanical pain threshold (PT) and expression of G protein-coupled receptor kinase (GRK5), ß-arrestin 2, total and phosphorylated PKC alpha (p-PKCα) proteins in the locus coeruleus (LC) of rats with bone cancer pain induced morphine tolerance, so as to reveal its partial central mechanisms underlying pain relief. METHODS: Forty SD rats were randomly divided into 5 groups, namely sham bone cancer, bone cancer pain, morphine tolerance, bone-edge EA, and sham EA (n＝ 8 rats in each group). The bone cancer with morphine tolerance model was established by intramedullary injection of MRMT-1 cells into the tibial cavity, and then intraperitoneal injection of morphine hydrochloride injection. After successful establishment of morphine tolerance model, the bone-edge EA (2 Hz/100 Hz，0.5－1.5 mA) was applied to bilateral "Zusanli" (ST36) and "Kunlun" (BL60) for 30 min, once a day for 7 days, after inserting the needle-tip to the tibial bone surface. The ipsilateral mechanical paw withdrawal thresholds (PWTs) were detected dynamically. The expression levels of GRK5, ß-arrestin 2, PKCα and p-PKCα in the LC area were measured by Western blot. RESULTS: The PWTs of bone cancer pain rats were decreased on day 10 after inoculation of cancer cells (P<0.01). After i．p. of morphine for 11 days, no analgesic effect and pain tolerance appeared (P>0.05). The PWTs were significantly increased in the bone-edge EA intervention group (P<0.01), not in the sham EA group (P>0.05). In comparison with the sham bone cancer group, the expression of GRK5 protein in morphine tolerance group was significantly decreased (P<0.01); compared with morphine tolerance group, the expression of GRK5 protein in bone-edge EA group was increased(P<0.01). In comparison with the sham bone cancer group, the expression of ß-arrestin 2 and p-PKCα in bone cancer group significantly increased (P<0.01). After the intervention, the increased ß-arrestin 2 and p-PKCα expressions were reversed in the bone-edge EA group (P<0.01); compared with morphine tolerance group and sham EA group, the expression of PKCα protein was decreased(P<0.01). CONCLUSION: Bone-edge EA can effectively relieve morphine tolerance in bone cancer pain rats, which may be related to its functions in up-regulating GRK5 protein and down-regulating ß-arrestin 2, PKCα and p-PKCα proteins in LC.　.

G Protein-Coupled Receptor Systems and Their Role in Cellular Senescence.

Aging is a complex biological process that is inevitable for nearly all organisms. Aging is the strongest risk factor for development of multiple neurodegenerative disorders, cancer and cardiovascular disorders. Age-related disease conditions are mainly caused by the progressive degradation of the integrity of communication systems within and between organs. This is in part mediated by, i) decreased efficiency of receptor signaling systems and ii) an increasing inability to cope with stress leading to apoptosis and cellular senescence. Cellular senescence is a natural process during embryonic development, more recently it has been shown to be also involved in the development of aging disorders and is now considered one of the major hallmarks of aging. G-protein-coupled receptors (GPCRs) comprise a superfamily of integral membrane receptors that are responsible for cell signaling events involved in nearly every physiological process. Recent advances in the molecular understanding of GPCR signaling complexity have expanded their therapeutic capacity tremendously. Emerging data now suggests the involvement of GPCRs and their associated proteins in the development of cellular senescence. With the proven efficacy of therapeutic GPCR targeting, it is reasonable to now consider GPCRs as potential platforms to control cellular senescence and the consequently, age-related disorders.

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 Systems as Crucial Regulators of DNA Damage Response Processes.

G protein-coupled receptors (GPCRs) and their associated proteins represent one of the most diverse cellular signaling systems involved in both physiological and pathophysiological processes. Aging represents perhaps the most complex biological process in humans and involves a progressive degradation of systemic integrity and physiological resilience. This is in part mediated by age-related aberrations in energy metabolism, mitochondrial function, protein folding and sorting, inflammatory activity and genomic stability. Indeed, an increased rate of unrepaired DNA damage is considered to be one of the 'hallmarks' of aging. Over the last two decades our appreciation of the complexity of GPCR signaling systems has expanded their functional signaling repertoire. One such example of this is the incipient role of GPCRs and GPCR-interacting proteins in DNA damage and repair mechanisms. Emerging data now suggest that GPCRs could function as stress sensors for intracellular damage, e.g., oxidative stress. Given this role of GPCRs in the DNA damage response process, coupled to the effective history of drug targeting of these receptors, this suggests that one important future activity of GPCR therapeutics is the rational control of DNA damage repair systems.

The Role of G Protein-coupled Receptor Kinases in Cancer.

G protein-coupled receptors (GPCRs) are the largest family of plasma membrane receptors. Emerging evidence demonstrates that signaling through GPCRs affects numerous aspects of cancer biology such as vascular remolding, invasion, and migration. Therefore, development of GPCR-targeted drugs could provide a new therapeutic strategy to treating a variety of cancers. G protein-coupled receptor kinases (GRKs) modulate GPCR signaling by interacting with the ligand-activated GPCR and phosphorylating its intracellular domain. This phosphorylation initiates receptor desensitization and internalization, which inhibits downstream signaling pathways related to cancer progression. GRKs can also regulate non-GPCR substrates, resulting in the modulation of a different set of pathophysiological pathways. In this review, we will discuss the role of GRKs in modulating cell signaling and cancer progression, as well as the therapeutic potential of targeting GRKs.

Quantitation of Plasma Membrane (G Protein-Coupled) Receptor Trafficking in Cultured Cells.

Measuring the functional behavior of G protein-coupled receptors (GPCRs) has been a major focus of academic and pharmaceutical research for many decades. These efforts have led to the development of many assays to measure the downstream effects of ligand binding on receptor activity. In this chapter, we describe an internalization/recycling assay that can be used to track changes in receptor number at the plasma membrane. Used in concert with other assays, this antibody-based technique can provide important information on GPCR activation by receptor-specific ligands.

Increased G protein-coupled receptor kinase (GRK) expression in the anterior cingulate cortex in schizophrenia.

BACKGROUND: Current pharmacological treatments for schizophrenia target G protein-coupled receptors (GPCRs), including dopamine receptors. Ligand-bound GPCRs are regulated by a family of G protein-coupled receptor kinases (GRKs), members of which uncouple the receptor from heterotrimeric G proteins, desensitize the receptor, and induce receptor internalization via the arrestin family of scaffolding and signaling molecules. GRKs initiate the activation of downstream signaling pathways, can regulate receptors and signaling molecules independent of GPCR phosphorylation, and modulate epigenetic regulators like histone deacetylases (HDACs). We hypothesize that the expression of GRK proteins is altered in schizophrenia, consistent with previous findings of alterations upstream and downstream from this family of molecules that facilitate intracellular signaling processes. METHODS: In this study, we measured protein expression via Western blot analysis for GRKs 2, 3, 5, and 6 in the anterior cingulate cortex of patients with schizophrenia (n=36) and a comparison group (n=33). To control for antipsychotic treatment, we measured these same targets in haloperidol-treated vs. untreated rats (n=10 for both). RESULTS: We found increased levels of GRK5 in schizophrenia. No changes were detected in GRK protein expression in rats treated with haloperidol decanoate for 9 months. CONCLUSION: These data suggest that increased GRK5 expression may contribute to the pathophysiology of schizophrenia via abnormal regulation of the cytoskeleton, endocytosis, signaling, GPCRs, and histone modification.