Feedback inhibition of G protein-coupled receptor kinase 2 (GRK2) activity by extracellular signal-regulated kinases

G protein-coupled receptor kinase (GRK)-mediated receptor phosphorylation and beta-arrestin binding uncouple G protein-coupled receptors (GPCRs) from their respective G proteins and initiates the process of receptor internalization. In the case of the beta(2)-adrenergic receptor and lysophosphatidic acid receptor, these processes can lead to ERK activation. Here we identify a novel mechanism whereby the activity of GRK2 is regulated by feedback inhibition. GRK2 is demonstrated to be a phosphoprotein in cells. Mass spectrometry and mutational analysis localize the site of phosphorylation on GRK2 to a carboxyl-terminal serine residue (Ser(670)). Phosphorylation at Ser(670) impairs the ability of GRK2 to phosphorylate both soluble and membrane-incorporated receptor substrates and dramatically attenuates Gbetagamma-mediated activation of this enzyme. Ser(670) is located in a peptide sequence that conforms to an ERK consensus phosphorylation sequence, and in vitro, in the presence of heparin, ERK1 phosphorylates GRK2. Inhibition of ERK activity in HEK293 cells potentiates GRK2 activity, whereas, conversely, ERK activation inhibits GRK2 activity. The discovery that ERK phosphorylates and inactivates GRK2 suggests that ERK participates in a feedback regulatory loop. By negatively regulating GRK-mediated receptor phosphorylation, beta-arrestin-mediated processes such as Src recruitment and clathrin-mediated internalization, which are required for GPCR-mediated ERK activation, are inhibited, thus dampening further ERK activation.     

Modulation of extracellular signal-regulated kinase (ERK) activity by acute and chronic opioid treatment in neuronal and glial cell lines

Acute mu opioid application has been shown to activate extracellular signal-related kinases (ERKs) in various non-neural cell lines. However, ERK activation in neuronal cells following acute morphine treatment is more questionable. Moreover, the ERK activation phenomenon observed in vivo after withdrawal of chronic opioids has never been demonstrated in vitro. The goal of this study was to determine if mu agonist treatment induced ERK activation acutely or after withdrawal of chronic opioids in one glial and three neuronal cell lines. We found that acute application of opioids was not able to activate ERK in neuronal cell lines but was able to activate ERK in a glial cell line. In another set of experiments, cells were chronically treated with escalating doses of a mu opioid agonist. After 8 days, the agonist was removed from the media and naloxone applied. Acute ERK activation was not seen in any tested cell line after agonist removal. These findings suggest that opioids may acutely activate ERK in non-neuronal cells, and that the acute ERK activation observed in some brain regions during opioid withdrawal in vivo might be mediated by indirect effects on neuronal cells.     

Integrins regulate microtubule nucleating activity of centrosome through mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase (MEK/ERK) signaling

Microtubule nucleation is an essential step in the formation of the microtubule cytoskeleton. We recently showed that androgen and Src promote microtubule nucleation and γ-tubulin accumulation at the centrosome. Here, we explore the mechanisms by which androgen and Src regulate these processes and ask whether integrins play a role. We perturb integrin function by a tyrosine-to-alanine substitution in membrane-proximal NPIY motif in the integrin β1 tail and show that this mutant substantially decreases microtubule nucleation and γ-tubulin accumulation at the centrosome. Because androgen stimulation promotes the interaction of the androgen receptor with Src, resulting in PI3K/AKT and MEK/ERK signaling, we asked whether these pathways are inhibited by the mutant integrin and whether they regulate microtubule nucleation. Our results indicate that the formation of the androgen receptor-Src complex and the activation of downstream pathways are significantly suppressed when cells are adhered by the mutant integrin. Inhibitor studies indicate that microtubule nucleation requires MEK/ERK but not PI3K/AKT signaling. Importantly, the expression of activated RAF-1 is sufficient to rescue microtubule nucleation inhibited by the mutant integrin by promoting the centrosomal accumulation of γ-tubulin. Our data define a novel paradigm of integrin signaling, where integrins regulate microtubule nucleation by promoting the formation of androgen receptor-Src signaling complexes to activate the MEK/ERK signaling pathway.     

Inhibition of mu and delta but not kappa opioid binding to membranes by Fab fragments from a monoclonal antibody directed against the opioid receptor

Fab fragments from a monoclonal antibody, OR-689.2.4, directed against the opioid receptor, selectively inhibited opioid binding to rat and guinea pig neural membranes. In a titratable manner, the Fab fragments noncompetitively inhibited the binding of the mu selective peptide [D-Ala2,(Me)Phe4,Gly(OH)5][3H] enkephalin and the delta selective peptide [D-Pen2,D-Pen5] [3H]enkephalin (where Pen represents penicillamine) to neural membranes. In contrast, kappa opioid binding, as measured by the binding of [3H]bremazocine to rat neural membranes and guinea pig cerebellum in the presence of mu and delta blockers, was not significantly altered by the Fab fragments. In addition to blocking the binding of mu and delta ligands, the Fab fragments displaced bound opioids from the membranes. When mu sites were blocked with [D-Ala2,(Me)Phe4,Gly(OH)5]enkephalin, the Fab fragments suppressed the binding of [D-Pen2,D-Pen5][3H]enkephalin to the same degree as when the mu binding site was not blocked. The Fab fragments also inhibited binding to the mu site regardless of whether or not the delta site was blocked with [D-Pen2,D-Pen5]enkephalin. This monoclonal antibody is directed against a 35,000-dalton protein. Since the antibody is able to inhibit mu and delta binding but not kappa opioid binding, it appears that this 35,000-dalton protein is an integral component of mu and delta opioid receptors but not kappa receptors.     

Effects of rho kinase and actin stress fibers on sustained extracellular signal-regulated kinase activity and activation of G(1) phase cyclin-dependent kinases

We recently reported that Rho kinase is required for sustained ERK signaling and the consequent mid-G(1) phase induction of cyclin D1 in fibroblasts. The results presented here indicate that these Rho kinase effects are mediated by the formation of stress fibers and the consequent clustering of alpha5beta1 integrin. Mechanistically, alpha5beta1 signaling and stress fiber formation allowed for the sustained activation of MEK, and this effect was mediated upstream of Ras-GTP loading. Interestingly, disruption of stress fibers with ML-7 led to G(1) phase arrest while comparable disruption of stress fibers with Y27632 (an inhibitor of Rho kinase) or dominant-negative Rho kinase led to a more rapid progression through G(1) phase. Inhibition of either MLCK or Rho kinase blocked sustained ERK signaling, but only Rho kinase inhibition allowed for the induction of cyclin D1 and activation of cdk4 via Rac/Cdc42. The levels of cyclin E, cdk2, and their major inhibitors, p21(cip1) and p27(kip1), were not affected by inhibition of MLCK or Rho kinase. Overall, our results indicate that Rho kinase-dependent stress fiber formation is required for sustained activation of the MEK/ERK pathway and the mid-G(1) phase induction of cyclin D1, but not for other aspects of cdk4 or cdk2 activation. They also emphasize that G(1) phase cell cycle progression in fibroblasts does not require stress fibers if Rac/Cdc42 signaling is allowed to induce cyclin D1.     

MAP kinase activation by mu opioid receptor involves phosphatidylinositol 3-kinase but not the cAMP/PKA pathway.

The involvement of protein kinases was studied in mu opioid receptor activation of mitogen-activated protein (MAP) kinase using cells transfected with the receptor clone. The cAMP/protein kinase A (PKA) pathway is known to be the major biochemical pathway for mu opioid receptor signaling. However, our data showed that stimulating adenylyl cyclase or activating PKA had no effect on mu receptor enhancement of MAP kinase activity, suggesting that the cAMP/PKA pathway is not involved in mediating the mu receptor activation of MAP kinase. Inhibition of phosphatidylinositol (PI) 3-kinase reduced mu receptor enhancement of MAP kinase activity, suggesting PI 3-kinase involvement. Together, these results show that cross-talk between the mu opioid receptor and the MAP kinase cascade is not mediated by the cAMP/PKA pathway, but involves PI 3-kinase.     

Phosphorylation of p90 ribosomal S6 kinase (RSK) regulates extracellular signal-regulated kinase docking and RSK activity

Stimulation of the Ras/extracellular signal-regulated kinase (ERK) pathway can modulate cell growth, proliferation, survival, and motility. The p90 ribosomal S6 kinases (RSKs) comprise a family of serine/threonine kinases that lie at the terminus of the ERK pathway. Efficient RSK activation by ERK requires its interaction through a docking site located near the C terminus of RSK, but the regulation of this interaction remains unknown. In this report we show that RSK1 and ERK1/2 form a complex in quiescent HEK293 cells that transiently dissociates upon mitogen stimulation. Complex dissociation requires phosphorylation of RSK1 serine 749, which is a mitogen-regulated phosphorylation site located near the ERK docking site. Using recombinant RSK1 proteins, we find that serine 749 is phosphorylated by the N-terminal kinase domain of RSK1 in vitro, suggesting that ERK1/2 dissociation is mediated through RSK1 autophosphorylation of this residue. Consistent with this hypothesis, we find that inactivating mutations in the RSK1 kinase domains disrupted the mitogen-regulated dissociation of ERK1/2 in vivo. Analysis of different RSK isoforms revealed that RSK1 and RSK2 readily dissociate from ERK1/2 following mitogen stimulation but that RSK3 remains associated with active ERK1/2. RSK activity assays revealed that RSK3 also remains active longer than RSK1 and RSK2, suggesting that prolonged ERK association increased the duration of RSK3 activation. These results provide new evidence for the regulated nature of ERK docking interactions and reveal important differences among the closely related RSK family members.     

Agonist-dependent desensitization of the kappa opioid receptor by G protein receptor kinase and beta-arrestin.

We used the Xenopus oocyte expression system to examine the regulation of rat kappa opioid receptor (rKOR) function by G protein receptor kinases (GRKs). kappa agonists increased the conductance of G protein-activated inwardly rectifying potassium channels in oocytes co-expressing KOR with Kir3.1 and Kir3.4. In the absence of added GRK and beta-arrestin 2, desensitization of the kappa agonist-induced potassium current was modest. Co-expression of either GRK3 or GRK5 along with beta-arrestin 2 significantly increased the rate of desensitization, whereas addition of either beta-arrestin 2, GRK3, or GRK5 alone had no effect on the KOR desensitization rate. The desensitization was homologous as co-expressed delta opioid receptor-evoked responses were not affected by KOR desensitization. The rate of GRK3/beta-arrestin 2-dependent desensitization was reduced by truncation of the C-terminal 26 amino acids, KOR(Q355Delta). In contrast, substitution of Ala for Ser within the third intracellular loop [KOR(S255A,S260A, S262A)] did not reduce the desensitization rate. Within the C-terminal region, KOR(S369A) substitution significantly attenuated desensitization, whereas the KOR(T363A) and KOR(S356A,T357A) point mutations did not. These results suggest that co-expression of GRK3 or GRK5 and beta-arrestin 2 produced homologous, agonist-induced desensitization of the kappa opioid receptor by a mechanism requiring the phosphorylation of the serine 369 of rKOR.     

5-hydroxytryptamine 4 receptor activation of the extracellular signal-regulated kinase pathway depends on Src activation but not on G protein or beta-arrestin signaling

The 5-hydroxytryptamine(4) (5-HT(4)) receptors have recently emerged as key modulators of learning, memory, and cognitive processes. In neurons, 5-hydroxytryptamine(4) receptors (5-HT(4)Rs) activate cAMP production and protein kinase A (PKA); however, nothing is known about their ability to activate another key signaling pathway involved in learning and memory: the extracellular signal-regulated kinase (ERK) pathway. Here, we show that 5-HT(4)R stimulation, in primary neurons, produced a potent but transient activation of the ERK pathway. Surprisingly, this activation was mostly PKA independent. Similarly, using pharmacological, genetic, and molecular tools, we observed that 5-HT(4)Rs in human embryonic kidney 293 cells, activated the ERK pathway in a G(s)/cAMP/PKA-independent manner. We also demonstrated that other classical G proteins (G(q)/G(i)/G(o)) and associated downstream messengers were not implicated in the 5-HT(4)R-activated ERK pathway. The 5-HT(4)R-mediated ERK activation seemed to be dependent on Src tyrosine kinase and yet totally independent of beta-arrestin. Immunocytofluorescence revealed that ERK activation by 5-HT(4)R was restrained to the plasma membrane, whereas p-Src colocalized with the receptor and carried on even after endocytosis. This phenomenon may result from a tight interaction between 5-HT(4)R and p-Src detected by coimmunoprecipitation. Finally, we confirmed that the main route by which 5-HT(4)Rs activate ERKs in neurons was Src dependent. Thus, in addition to classical cAMP/PKA signaling pathways, 5-HT(4)Rs may use ERK pathways to control memory process.     

EBP50 exerts tumor suppressor activity by promoting cell apoptosis and retarding extracellular signal-regulated kinase activity

The expression of Ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) and the intragenic mutation of the ebp50 gene have been reported to correlate with human breast cancer development, but the exact impacts on breast cancer development and its molecular mechanism are not fully understood. In this study, we investigate the potential function of EBP50 through over-expression in the breast cancer cell line, MDA-MB-231, which has low EBP50 protein expression levels. The effects of EBP50 over-expression on cellular proliferation, anchorage-independent growth and apoptosis were examined. In addition, the activity of extracellular signal-regulated kinase (ERK) was also determined. Our results show that a decrease of cellular proliferation and attenuation of colony-forming ability were evident in MDA-MB-231 cells stably transfected with an EBP50 expressing plasmid (EBP-231) when compared with control cells. There was also a statistically significant increase in spontaneous apoptosis in EBP-231 cells accompanied by an attenuation in ERK activity. Altogether, our results suggest that restoring EBP50 expression could suppress breast cancer cell proliferation by promoting cell apoptosis and inhibiting ERK activity, and that EBP50 may be a target for development of diagnostics and therapeutics in breast cancer.     

Induction of PC12 cell differentiation by flavonoids is dependent upon extracellular signal-regulated kinase activation

Many of the physiological benefits attributed to flavonoids are thought to stem from their potent antioxidant and free radical scavenging properties. Recently, it was shown that flavonoids protect nerve cells from oxidative stress by multiple mechanisms, only one of which is directly related to their antioxidant activity, suggesting that specific flavonoids may have other properties that could make them useful in the treatment of conditions that lead to nerve cell death. In particular, it was asked if any flavonoid could mimic neurotrophic proteins. To examine this possibility, we looked at the ability of flavonoids to induce nerve cell differentiation using PC12 cells. PC12 cells were treated with a variety of flavonoids to determine if there was a correlation between their neuroprotective activity and their neurite outgrowth-promoting activity. In addition, the signaling pathways required for flavonoid-induced differentiation were examined. We found that only a small subset of the flavonoids that were neuroprotective could induce neurite outgrowth by an extracellular signal-regulated kinase-dependent process. There was a strong correlation between the concentrations of the flavonoids that were neuroprotective and the concentrations that induced differentiation. These results suggest that the consumption of specific flavonoids could have further beneficial effects on nerve cells following injury, in pathological conditions or in normal aging.     

Activation of extracellular signal-regulated kinase by 12-hydroxyheptadecatrienoic acid in prostate cancer PC3 cells

Both 12-hydroxyheptadecatrienoic acid (12-HHT) and thromboxane A₂ (TXA₂) are products derived from prostaglandin H₂ (PGH₂) catalyzed by thromboxane synthase. Whether or not they exhibit similar actions remains to be determined. While TXA₂-induced activation of extracellular signal-regulated kinases (ERKs) has been extensively studied, 12-HHT-induced activation of ERKs has not been explored. We reported for the first time that 12-HHT induced activation of ERKs in human prostate cancer cell line, PC3. We also compared the mechanisms of 12-HHT- and I-BOP-, a TXA₂ mimetic, mediated ERK activation in PC3 cells. The activation of ERKs induced by either agent was shown to involve protein kinase C (PKC)-, protein kinase A (PKA)-, Src kinase and phosphoinositide-3 kinase (PI-3K)-dependent mechanisms in addition to the transactivation of the EGF receptor (EGFR) and the involvement of matrix metalloproteinases (MMPs) based on the sensitivity of the activation to their respective inhibitors. JNK/SAPK and p38 MAPK pathways were responsive to I-BOP but not to 12-HHT stimulation. Both 12-HHT- and I-BOP-induced activations of ERKs were also examined in other human prostate cancer cells, human lung cancer cells, and human lung fibroblast. I-BOP appeared to induce activation of ERKs in most cell lines, whereas 12-HHT induced activation of ERKs only in lung fibroblast in addition to PC3 cells. It appears that TPs are more generally expressed and the potential 12-HHT receptor (s) is expressed in limited specific cell types. Our results suggest that increased expression of thromboxane synthase as seen in prostate tumor may stimulate tumorigenesis as a consequence of concurrent increased synthesis of two fatty acids capable of activating ERKs.     

Expression of delta, kappa and mu human opioid receptors in Escherichia coli and reconstitution of the high-affinity state for agonist with heterotrimeric G proteins.

Human opioid receptors of the delta, mu and kappa subtypes were successfully expressed in Escherichia coli as fusions to the C-terminus of the periplasmic maltose-binding protein, MBP. Expression levels of correctly folded receptor molecules were comparable for the three subtypes and reached an average of 30 receptors.cell-1 or 0.5 pmol.mg-1 membrane protein. Binding of [3H]diprenorphine to intact cells or membrane preparations was saturatable, with a dissociation constant, KD, of 2.5 nM, 0.66 nM and 0.75 nM for human delta, mu and kappa opioid receptors (hDOR, hMOR and hKOR, respectively). Recombinant receptors of the three subtypes retained selectivity and nanomolar affinity for their specific antagonists. Agonist affinities were decreased by one to three orders of magnitude as compared to values measured for receptors expressed in mammalian cells. The effect of sodium on agonist binding to E. coli-expressed receptors was investigated. Receptor high-affinity state for agonists was reconstituted in the presence of heterotrimeric G proteins. We also report affinity values of endomorphins 1 and 2 for mu opioid receptors expressed both in E. coli and in COS cells. Our results confirm that opioid receptors can be expressed in a functional form in bacteria and point out the advantages of E. coli as an expression system for pharmacological studies.     

Differential modulation of nociceptive responses to mu and kappa opioid receptor directed drugs by blood glucose in experimentally induced diabetes rats

The study has evaluated the effect of diabetes associated hyperglycaemia on nociception and antinociception induced by morphine, buprenorphine and pentazocine in female albino rats. Rats were allocated into 3 groups of 20 each--group I consisted of control having normal blood glucose levels (BGLs), group II consisted of streptozotocin-induced diabetics (STZ-D) having hyperglycaemia and group III consisted of diabetic rats controlled with insulin treatment. Immediately before and 15, 30 min, 1, 2 and 3 hr after injection with test drugs, rats were subjected to a thermal noxious stimulus using tail withdrawal from hot water and tail-flick latencies (TFL) so generated were recorded. Similarly, before and 30, 45 min and 1 hr after injection with drugs rats were subjected to abdominal writhing with hypertonic saline and number of writhes were counted per 90 sec. In STZ-D animals (BGLs 317.95 +/- 3.8 mg/dl) a decreased TFL with an increase in the number of writhes compared to control and diabetes controlled with insulin treatment was observed. Percent maximum possible effect of morphine (5 mg/kg, s.c.) and buprenorphine (2 mg/kg, s.c.) was significantly lower when compared to control as well as STZ-D controlled with insulin treatment groups. Similarly percent protection from writhing of morphine (0.05 mg/kg, s.c.) and buprenorphine (0.01 mg/kg, s.c.) was significantly less in comparison to control and STZ-D controlled with insulin treatment group. However, percent maximum possible effect of pentazocine (20 mg/kg, s.c.) and percent protection from writhing of pentazocine (1 mg/kg, s.c.) was significantly high in STZ-D rats when compared to control and STZ-D rats controlled with insulin treatment groups. The results suggest that both mu and kappa--opioid receptors may be modulated by blood glucose levels possibly involving cellular energetics mediated change in potassium (KATP) channels in females rats, albeit differentially.     

Altered extracellular signal-regulated kinase signal transduction by the muscarinic acetylcholine and metabotropic glutamate receptors after cerebral ischemia.

To determine whether muscarinic acetylcholine receptors (mAChR) in the post-ischemic hippocampus may be involved in altered extracellular signal-regulated kinases (ERK) signal transduction, we have investigated changes in the activity of ERK1/2 induced by a muscarinic agonist, carbachol. Cerebral ischemia was produced in the rat by injecting 900 microspheres (48 microm in diameter) into the right internal carotid artery. Applying carbachol to the contralateral hippocampal slices from ischemic rats increased the phosphorylation of ERK1/2 but did not increase phosphorylation in the ipsilateral hippocampus. Analysis of M(1) mAChR binding showed that there was no significant difference in the number and K(d) values between the hippocampi from na?ve and ischemic rats. Immunoblotting analysis showed no significant difference in the amount of M(1) mAChR in both hemispheres. In contrast to carbachol stimulation, the protein kinase C activator induced an activation of ERK1/2 in the ipsilateral hippocampus. This increase was shown to occur in neurons by immunofluorescence colocalization study. Carbachol-stimulated tyrosine phosphorylation of the G alpha(q/11), inositol 1,4,5-trisphosphate formation, and association of G alpha(q/11) with phospholipase C beta 1 were attenuated in the ipsilateral hippocampus. We also found that stimulation of group I metabotropic glutamate receptors, which are linked to G alpha(q/11), failed to increase in phosphorylation of ERK1/2 in the ipsilateral hippocampus. These results suggest that failure in receptor-mediated tyrosine phosphorylation of the G alpha(q/11) subunit and a defect in receptor-G alpha(q/11-)effector coupling in the ischemic hippocampus may be involved in alterations of ERK signal transduction.     

A novel function of Goalpha: mediation of extracellular signal-regulated kinase activation by opioid receptors in neural cells

Go is the most abundant G protein expressed in brain but its function is less known. Here we show a novel function of Goalpha as a mediator of opioid receptor-induced extracellular signal-regulated kinase activation in neural cells. The current study found that, in neuroblastoma x glioma NG108-15 hybrid cells, activation of extracellular signal-regulated kinase through delta opioid receptors was mediated by pertussis toxin-sensitive G protein and independent of Gbetagamma subunits, PI3 kinase and receptor internalization. Overexpression of a dominant negative form of Goalpha1, but not Gialpha2, completely blocked delta opioid receptor-induced extracellular signal-regulated kinase activity. Decreasing Goalpha expression by RNA interference greatly reduced delta opioid receptor-induced extracellular signal-regulated kinase activity and extracellular signal-regulated kinase-dependent gene expression, while knocking down Gialpha2 did not. By taking advantage of differences between human and mouse Goalpha gene sequences, we simultaneously knocked down endogenous Goalpha expression and expressed exogenous human Goalpha subunits. We found that both human Goalpha1 and Goalpha2 could mediate delta opioid receptor-induced extracellular signal-regulated kinase activation. This study suggests that one of the functions of Goalpha in the brain is to mediate extracellular signal-regulated kinase activation by G protein-coupled receptors.