Inhibition of G Protein-coupled Receptor Kinase Subtypes by Ca2+/Calmodulin

G protein-coupled receptor kinases (GRKs) are implicated in the homologous desensitization of G protein-coupled receptors. Six GRK subtypes have so far been identified, named GRK1 to GRK6. The functional state of the GRKs can be actively regulated in different ways. In particular, it was found that retinal rhodopsin kinase (GRK1), but not the ubiquitous betaARK1 (GRK2), can be inhibited by the photoreceptor-specific Ca2+-binding protein recoverin through direct binding. The present study was aimed to investigate regulation of other GRKs by alternative Ca2+-binding proteins such as calmodulin (CaM). We found that Gbetagamma-activated GRK2 and GRK3 were inhibited by CaM to similar extents (IC50 approximately 2 microM), while a 50-fold more potent inhibitory effect was observed on GRK5 (IC50 = 40 nM). Inhibition by CaM was strictly dependent on Ca2+ and was prevented by the CaM inhibitor CaMBd. Since Gbetagamma, which is a binding target of Ca2+/CaM, is critical for the activation of GRK2 and GRK3, it provides a possible site of interaction between these proteins. However, since GRK5 is Gbetagamma-independent, an alternative mechanism is conceivable. A direct interaction between GRK5 and Ca2+/CaM was revealed using CaM-conjugated Sepharose 4B. This binding does not influence the catalytic activity as demonstrated using the soluble GRK substrate casein. Instead, Ca2+/CaM significantly reduced GRK5 binding to the membrane. The mechanism of GRK5 inhibition appeared to be through direct binding to Ca2+/CaM, resulting in inhibition of membrane association and hence receptor phosphorylation. The present study provides the first evidence for a regulatory effect of Ca2+/CaM on some GRK subtypes, thus expanding the range of different mechanisms regulating the functional states of these kinases

High expression of beta-adrenergic receptor kinase in human peripheral blood leukocytes. Isoproterenol and platelet activating factor can induce kinase translocation.

Receptor phosphorylation is a key step in the process of desensitization of the beta-adrenergic and other related receptors. A selective kinase (called beta-adrenergic receptor kinase, beta ARK) has been identified which phosphorylates the agonist-occupied form of the receptor. Recently the bovine beta ARK cDNA has been cloned and the highest levels of specific mRNA were found in highly innervated tissues. It was proposed that beta ARK may be primarily active on synaptic receptors. In the present study, the cDNA of human beta ARK was cloned and sequenced. The sequence was very similar to that of the bovine beta ARK (the overall amino acid homology was 98%). Very high levels of beta ARK mRNA and kinase activity were found in peripheral blood leukocytes and in several myeloid and lymphoid leukemia cell lines. Since agonist-induced beta ARK translocation is considered the first step involved in beta ARK-mediated homologous desensitization, we screened a number of G-protein-coupled receptor agonists for their ability to induce beta ARK translocation. In human mononuclear leukocytes, beta-AR agonist isoproterenol and platelet-activating factor were able to induce translocation of beta ARK from cytosol to membrane. After 20 min of exposure to isoproterenol (10 microM), the cytosolic beta ARK activity decreased to 61% of control, while membrane-associated beta ARK activity increased to 170%. 20-min exposure to platelet-activating factor (1 microM) reduced the cytosolic beta ARK activity to 42% of control with concomitant increase in membrane beta ARK activity to 214% of control. The high levels of beta ARK expression in human peripheral blood leukocytes together with the ability of isoproterenol and platelet-activating factor to induce beta ARK translocation, suggest a role for beta ARK in modulating some receptor-mediated immune functions

The ε3 and ε4 Alleles of Human APOE Differentially Affect Tau Phosphorylation in Hyperinsulinemic and Pioglitazone Treated Mice

Impaired insulin signalling is increasingly thought to contribute to Alzheimer's disease (AD). The ε4 isoform of the APOE gene is the greatest genetic risk factor for sporadic, late onset AD, and is also associated with risk for type 2 diabetes mellitus (T2DM). Neuropathological studies reported the highest number of AD lesions in brain tissue of ε4 diabetic patients. However other studies assessing AD pathology amongst the diabetic population have produced conflicting reports and have failed to show an increase in AD-related pathology in diabetic brain. The thiazolidinediones (TZDs), peroxisome proliferator-activated receptor gamma agonists, are peripheral insulin sensitisers used to treat T2DM. The TZD, pioglitazone, improved memory and cognitive functions in mild to moderate AD patients. Since it is not yet clear how apoE isoforms influence the development of T2DM and its progression to AD, we investigated amyloid beta and tau pathology in APOE knockout mice, carrying human APOEε3 or ε4 transgenes after diet-induced insulin resistance with and without pioglitazone treatment.Male APOE knockout, APOEε3-transgenic and APOEε4-transgenic mice, together with background strain C57BL6 mice were kept on a high fat diet (HFD) or low fat diet (LFD) for 32 weeks, or were all fed HFD for 32 weeks and during the final 3 weeks animals were treated with pioglitazone or vehicle.All HFD animals developed hyperglycaemia with elevated plasma insulin. Tau phosphorylation was reduced at 3 epitopes (Ser396, Ser202/Thr205 and Thr231) in all HFD, compared to LFD, animals independent of APOE genotype. The introduction of pioglitazone to HFD animals led to a significant reduction in tau phosphorylation at the Ser202/Thr205 epitope in APOEε3 animals only. We found no changes in APP processing however the levels of soluble amyloid beta 40 was reduced in APOE knockout animals treated with pioglitazone

Phosphorylation and Activation of β-Adrenergic Receptor Kinase by Protein Kinase C

The aim of this study was to test the possible modification of beta-adrenergic receptor kinase (beta ARK) activity by second messengers and/or their downstream components. Using human mononuclear leukocytes (MNL), we found that calcium ionophores could elevate beta ARK activity by about 80% in a protein kinase C (PKC)-dependent manner. This was confirmed by the ability of the PKC activator phorbol 12-myristate 13-acetate (PMA) to produce a similar effect, suggesting a PKC-dependent modulation of beta ARK activity. In vitro experiments with purified proteins showed that PKC could directly phosphorylate beta ARK1 with an apparent Km for beta ARK1 of 6 nM. The ability of beta ARK1 to phosphorylate rhodopsin was 61% greater when it was phosphorylated by PKC. The level of phosphorylation of beta ARK1 immunoprecipitated from MNL and Sf9 cells overexpressing this kinase was enhanced by about 2-3-fold after PMA treatment. Functional significance of PKC-dependent increase in beta ARK activity ws demonstrated by beta-adrenergic receptor (beta AR) homologous desensitization experiments in MNL. beta AR desensitization, as induced by exposure to 10 microM isoproterenol (5 min at 37 degrees C), was increased from 42 +/- 10% in control to 68 +/- 8% in PMA-pretreated MNL. beta ARK inhibitor heparin (160 micrograms/ml) prevented the augmenting effect of PMA on beta AR desensitization. These results show that beta ARK activity can be increased through phosphorylation by PKC, thus indicating that beta ARK can be preconditioned to modulate the subsequent cellular responsiveness to receptor activation, providing the cell with a mechanism by which specific homologous desensitization can be regulated heterologously

G protein-coupled receptors: Heterologous regulation of homologous desensitization and its implications

Two patterns of rapid desensitization have been characterized for G protein-coupled receptn:d: homologous desensitizeton, which mainly involves G protein-coupled receptor kineses and arrestins, and heterologous desensitization, which mainly involves protein kinases A IPKA) and C (PKC). In this review, Tsu T&en Chuang and colleagues discuss evidence to show that PKA and PKC can modify the functional state of the G protein-coupled receptor kinases/arrestin homologous desensitization machinery, providing a novel level of cross-talk in signal transduction. Studies on regulation of G protein-coupled receptor kinases and arrestins confirm that the functional state of this machinery may have important consequences far cellular responsiveness and may represent new targets for therapeutic strategies

Identification of a short sequence highly divergent between beta-adrenergic-receptor kinases 1 and 2 that determines the affinity of binding to beta gamma subunits of heterotrimeric guanine-nucleotide-binding regulatory proteins

A 28-residue peptide (peptide G), derived from the C-terminal (W643-S670) of the beta-adrenergic receptor kinase (beta ARK), was previously identified as the critical domain for binding to the beta gamma subunits fo the heterotrimeric guanine-nucleotide-binding regulatory protein (G beta gamma). We observed that the 18-amino-acid core of this domain is poorly conserved between beta ARK1 and beta ARK2 and so may provide the basis for differences in G beta gamma-binding properties. Specific antibodies raised against 18-residue peptides derived from the divergent sequences (peptides P1 and P2 for beta ARK1 and beta ARK2, respectively) competitively inhibited G beta gamma-activation of the related beta ARK subtype, confirming the involvement of this region in binding to G beta gamma. Peptides P1 and P2 inhibited G beta gamma-stimulated activity of both beta ARK1 and beta ARK2, with P2 being significantly more potent than P1 (IC50 of 179 +/- 5 mu M for P2 and > 500 mu M for P1). The 28-residue peptides G showed the same relative inhibitory activities (IC50 = 48 +/- 5 mu M for G2 and 146 +/- 8 mu M for G1). This relative order of potency G2 > G1 approximate to P2 > P1 was confirmed in a direct G beta gamma-binding assay. No binding selectivity for the beta 1, beta 2, beta 3 and beta 4 G beta subtypes was observed. The EC50 value for G beta gamma-activation of beta ARK1 was about double of that for beta ARK2, indicating a higher affinity between G beta gamma and beta ARK2, which is the expected result based on the findings with the peptides. These findings show that the 18-residue peptides P represent the shortest sequence of beta ARK that can bind to G beta gamma and provide a demonstration of a functional difference between the G beta gamma binding domains of beta ARK1 and beta ARK2

Body weight increases in HFD fed mice.

<p>Body weight of APOE KO, APOEε3, APOEε4 and WT mice over 32 weeks of A) LFD and B) HFD, starting from 3 months of age. Values are mean±SEM, n = 10-12. Body weights in A) and B) reached ***p<0.001 by t-test at all weeks indicated compared to WT mice. Significance is indicated by * APOE KO, ⁺ APOEε3, ∧ APOEε4.</p

Levels of soluble Aβ40 and Aβ42 in HFD fed mice treated with or without pioglitazone.

<p>Aβ A) x-40 and B) x-42 was analysed by ELISA in the frontal cortex of APOE mice fed HFD and with or without pioglitazone treatment. *p<0.05 by t-test.</p