βArrestins

In this study we investigated the mechanisms responsible for MAP kinase ERK1/2 activation following agonist activation of endogenous mu opioid receptors (MOR) normally expressed in cultured striatal neurons. Treatment with the MOR agonist fentanyl caused significant activation of ERK1/2 in neurons derived from wild type mice. Fentanyl effects were blocked by the opioid antagonist naloxone and were not evident in neurons derived from MOR knock-out (؊/؊) mice. In contrast, ERK1/2 activation by fentanyl was not evident in neurons from GRK3 ؊/؊ mice or neurons pretreated with small inhibitory RNA for arrestin3. Consistent with this observation, treatment with the opiate morphine (which is less able to activate arrestin) did not elicit ERK1/2 activation in wild type neurons; however, transfection of arrestin3-(R170E) (a dominant positive form of arrestin that does not require receptor phosphorylation for activation) enabled morphine activation of ERK1/2. In addition, activation of ERK1/2 by fentanyl and morphine was rescued in GRK3 ؊/؊ neurons following transfection with dominant positive arrestin3-(R170E). The activation of ERK1/2 appeared to be selective as p38 MAP kinase activation was not increased by either fentanyl or morphine treatment in neurons from wild type, MOR ؊/؊ , or GRK3 ؊/؊ mice. In addition, U0126 (a selective inhibitor of MEK kinase responsible for ERK phosphorylation) blocked ERK1/2 activation by fentanyl. These results support the hypothesis that MOR activation of ERK1/2 requires opioid receptor phosphorylation by GRK3 and association of arrestin3 to initiate the cascade resulting in ERK1/2 phosphorylation in striatal neurons.Opioid receptor activation results in both acute and longlasting changes in neuronal physiology. The mechanisms responsible for the acute changes include G␣ i/o and G␤␥ protein activation that increases potassium conductance, decreases calcium conductance, and results in presynaptic inhibition (1-3). The mechanisms responsible for the long lasting effects of opioids are less clear but may include changes in adenylyl cyclase activity and activation of mitogen activating protein kinase (MAPK) 2 pathways (4, 5). In this study we used primary cultured neurons isolated from mouse striata to address the mechanisms linking mu opioid receptor (MOR) activation to the phosphorylation and activation of the extracellular signal-related kinases 1 and 2 (ERK1/2) members of the MAPK family. Phosphorylation of ERK1/2 by GPCRs involves growth factor receptor transactivation (4). As demonstrated for the ␤-adrenergic receptor, participation of arrestin is an integral part of GPCR signaling through the ERK1/2 signaling pathway in transfected HEK293 cells (6). Following agonist activation of MOR, the receptor becomes phosphorylated by G-protein receptor kinase (GRK), which initiates an arrestin-dependent desensitization process that involves clathrin-mediated endocytosis (7). The opioid activation of ERK1/2 by MOR agonist (D-Ala 2 ,Me-Phe 4 ,Gly-ol5) (DAMGO) was demonstrated in MOR-transfected c...

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Mu Opioid Receptor Activation of ERK1/2 Is GRK3 and Arrestin Dependent in Striatal Neurons

Macey

Lowe

Chavkin

2006

“…have been shown to act as molecular scaffolds for a variety of proteins (5)(6)(7)43). Analysis of the ␤-arrestin 1 crystal structure (44) shows it to be composed of two major regions, known as the N-and C-domains, each of which is composed of a seven-strand ␤-sandwich (Fig.…”

Section: Identification Of Two Regions In ␤-Arrestin That Are Involvementioning

confidence: 99%

The Unique Amino-terminal Region of the PDE4D5 cAMP Phosphodiesterase Isoform Confers Preferential Interaction with β-Arrestins

Bolger

McCahill

Huston

et al. 2003

101

139

Isoproterenol challenge of Hek-B2 cells causes a transient recruitment of the endogenous PDE4D isoforms found in these cells, namely PDE4D3 and PDE4D5, to the membrane fraction. PDE4D5 provides around 80% of the total PDE4D protein so recruited, although it only comprises about 40% of the total PDE4D protein in Hek-B2 cells. PDE4D5 provides about 80% of the total PDE4D protein found associated with ␤-arrestins immunopurified from Hek-B2, COS1, and A549 cells as well as cardiac myocytes, whereas its overall level in these cells is between 15 and 50% of the total PDE4D protein. Truncation analyses indicate that two sites in PDE4D5 are involved in mediating its interaction with ␤-arrestins, one associated with the common PDE4 catalytic region and the other located within its unique amino-terminal region. Truncation analyses indicate that two sites in ␤-arrestin 2 are involved in mediating its interaction with PDE4D5, one associated with its extreme amino-terminal region and the other located within the carboxylterminal domain of the protein. We suggest that the unique amino-terminal region of PDE4D5 allows it to preferentially interact with ␤-arrestins. This specificity appears likely to account for the preferential recruitment of PDE4D5, compared with PDE4D3, to membranes of Hek-B2 cells and cardiac myocytes upon challenge with isoproterenol.The heptahelical ␤-adrenergic receptors (␤ 2 ARs) 1 act by coupling through the G-protein G s to stimulate adenylyl cyclase and thereby increase intracellular cAMP concentrations (1-4). It is now well established that the rapid uncoupling of this response is achieved by the action of the G-protein receptorcoupled kinase 2 (2). This phosphorylates the carboxyl-terminal tail of the plasma membrane-associated ␤ 2 AR, allowing the recruitment of ␤-arrestins from the cytosol (5-7). It is this recruitment of ␤-arrestin, rather than phosphorylation per se, that elicits uncoupling, presumably by sterically blocking coupling of the ␤ 2 AR to G s . The importance of ␤-arrestin interaction to G-protein-coupled receptors (GPCRs) in vivo has been clearly established in knockout mouse models (8 -10).The attenuation of cAMP signaling is also achieved through the action of phosphodiesterases (PDEs) that are able to hydrolyze cAMP to 5Ј-AMP (11-17). Since they provide the sole route for degradation of cAMP in cells, they are poised to play a key role in controlling cAMP signaling. Multiple genes encode a large superfamily of PDEs, which differ in their regulatory and kinetic properties. Of these, the PDE4 cAMP-specific phosphodiesterase family (13-16) has recently attracted much interest, since PDE4-selective inhibitors are currently being developed as potential therapeutic agents for various inflammatory diseases of the respiratory system, such as asthma and chronic obstructive pulmonary disease (18 -21). The PDE4 enzyme family is encoded by four genes (PDE4A, -B, -C, and -D), which generate over 16 different isoforms through the use of distinct promoters and alternative mRNA splicing (12,13,...

“…It is worth pointing out that, for the A 2A -adenosine receptor, this was also true not only in CHO cells (where the receptor had been introduced by stable transfection) but in PC12 cells, which endogenously express the receptor. With the ␤ 2 -adrenergic receptor, ␤-arrestin serves as a docking site for SRC (10), and SRC-dependent phosphorylation of dynamin is essential for MAP kinase activation (38). It is evident that these mechanisms of recruiting are restricted to receptors that can directly bind SRC-like kinases or that require dynamin-dependent internalization.…”

Section: Blockage Of Src Family Kinases or Ablation Of Src Blunts Mapmentioning

confidence: 99%

“…Here ␤-arrestin functions as adapter protein for the recruitment of SRC and for the assembly of a large signaling complex. This model has been primarily developed with the ␤ 2 -adrenergic receptor (10) and predicts that stimulation of MAP kinase by the receptor is blocked by abrogating dynamin and SRC. (iv) Finally, G protein-coupled receptors may promote transactivation of tyrosine kinase receptors by causing the shedding of matrix-bound growth factors; this effect depends on the activation of matrix metalloproteases (11).…”

mentioning

confidence: 99%

MAP Kinase Stimulation by cAMP Does Not Require RAP1 but SRC Family Kinases

Klinger¹,

Kudlacek

Seidel³

et al. 2002

‫؍‬ 50 nM); this was also seen in PC12 cells, which express the A 2A -receptor endogenously, and in NIH3T3 fibroblasts, in which cAMP causes MAP kinase stimulation. In the corresponding murine fibroblast cell line SYF, which lacks the ubiquitously expressed SRC family kinases SRC, YES, and FYN, forskolin barely stimulated MAP kinase; this reduction was reversed in cells in which c-SRC had been reintroduced. These findings show that activation of MAP kinase by cAMP requires a SRC family kinase that lies downstream of protein kinase A. A role for RAP1, as documented for the ␤ 2 -adrenergic receptor, is apparently contingent on receptor endocytosis.Elevation of cyclic AMP inhibits the growth of many cells. This effect is thought to reflect, at least in part, the ability of protein kinase A (PKA) 1 -dependent phosphorylation to disrupt the interaction between p21 ras and c-RAF; this results in cAMP-mediated suppression of mitogen-activated protein kinase pathway (1-4). However, in some cells, agonist occupancy of G s -coupled receptors is associated with both increases in cellular cAMP and with stimulation of MAP kinase. It has been argued that stimulation of MAP kinase is dependent on cAMP and that effectors other than adenylyl cyclase generate the signal that link G s -coupled receptor to MAP kinase activation. Furthermore, in each of the proposed models, the signal to MAP kinase diverges at a different level from the signaling cascade composed of receptor, G s , and adenylyl cyclase/cAMP. (i) The cAMP-dependent signaling mechanism is mediated by the p21 ras -related, monomeric G protein RAP1 that preferentially activates B-RAF (5, 6). Loading of RAP1A with GTP requires guanine nucleotide exchange factors, a class of which (Epac) is directly activated by cAMP, i.e. in a manner independent of PKA (7,8). (ii) Alternatively, a role has been invoked for the non-receptor tyrosine kinase SRC, because G␣ s directly binds to and activates SRC in vitro (9). In this case, stimulation of MAP kinase by G s -coupled receptors is independent of cAMP but sensitive to inhibition or genetic ablation of SRC. (iii) A large set of experiments support a third model of MAP kinase activation in which the receptor is endocytosed in a dynamindependent fashion. Here ␤-arrestin functions as adapter protein for the recruitment of SRC and for the assembly of a large signaling complex. This model has been primarily developed with the ␤ 2 -adrenergic receptor (10) and predicts that stimulation of MAP kinase by the receptor is blocked by abrogating dynamin and SRC. (iv) Finally, G protein-coupled receptors may promote transactivation of tyrosine kinase receptors by causing the shedding of matrix-bound growth factors; this effect depends on the activation of matrix metalloproteases (11).Although G protein-coupled receptors may recruit multiple and redundant pathways to stimulate MAP kinase (12), it is evident that some of the proposed mechanisms are mutually exclusive. It is also difficult to understand why protein kinase A is required for cAMP-d...