G-protein receptor kinase 3 (GRK3) influences opioid analgesic tolerance but not opioid withdrawal.

1. Tolerance to opioids frequently follows repeated drug administration and affects the clinical utility of these analgesics. Studies in simple cellular systems have demonstrated that prolonged activation of opioid receptors produces homologous receptor desensitization by G-protein receptor kinase mediated receptor phosphorylation and subsequent beta-arrestin binding.

Increased acute inflammation, leukotriene B4-induced chemotaxis, and signaling in mice deficient for G protein-coupled receptor kinase 6.

Directed migration of polymorphonuclear neutrophils (PMN) is required for adequate host defense against invading organisms and leukotriene B(4) (LTB(4)) is one of the most potent PMN chemoattractants. LTB(4) exerts its action via binding to BLT1, a G protein-coupled receptor. G protein-coupled receptors are phosphorylated by G protein-coupled receptor kinases (GRK) in an agonist-dependent manner, resulting in receptor desensitization.

Enhanced rewarding properties of morphine, but not cocaine, in beta(arrestin)-2 knock-out mice.

The reinforcing and psychomotor effects of morphine involve opiate stimulation of the dopaminergic system via activation of mu-opioid receptors (muOR). Both mu-opioid and dopamine receptors are members of the G-protein-coupled receptor (GPCR) family of proteins. GPCRs are known to undergo desensitization involving phosphorylation of the receptor and the subsequent binding of beta(arrestins), which prevents further receptor-G-protein coupling.

Prolonged kappa opioid receptor phosphorylation mediated by G-protein receptor kinase underlies sustained analgesic tolerance.

Kappa opioid receptor (KOR) desensitization was previously shown to follow agonist-dependent phosphorylation of serine 369 by G-protein receptor kinase (GRK) and beta-arrestin binding in transfected cells. To study the in vivo effects induced by phosphorylation of KOR(S369), C57Bl/6 mice were administered single or repeated doses of the KOR agonist, U50,488, and isolated brain glycoprotein was probed with an antibody, KOR-P, that specifically recognized phosphoserine 369 KOR. Western blot analysis using KOR-P antibody showed that labeling intensity increased after either single or repeated treatment of mice with U50,488 by 59 +/- 22% and 101 +/- 29%, respectively.

G protein-coupled receptor kinase 5 regulates airway responses induced by muscarinic receptor activation.

G protein-coupled receptors (GPCRs) transduce extracellular signals into intracellular events. The waning responsiveness of GPCRs in the face of persistent agonist stimulation, or desensitization, is a necessary event that ensures physiological homeostasis. GPCR kinases (GRKs) are important regulators of GPCR desensitization.

Beta-arrestin1 mediates insulin-like growth factor 1 (IGF-1) activation of phosphatidylinositol 3-kinase (PI3K) and anti-apoptosis.

beta-arrestins (1 and 2) are widely expressed cytosolic proteins that play central roles in G protein-coupled receptor signaling. beta-arrestin1 is also recruited to the insulin-like growth factor 1 (IGF-1) receptor, a receptor tyrosine kinase, upon agonist binding. Here we report that, in response to IGF-1 stimulation, beta-arrestin1 mediates activation of phosphatidylinositol 3-kinase in a pathway that leads to the subsequent activation of Akt and anti-apoptosis.

Regulation of V2 vasopressin receptor degradation by agonist-promoted ubiquitination.

The seven-transmembrane-spanning vasopressin V2 receptor (V2R) is a Gs-coupled receptor that is rapidly phosphorylated and internalized following stimulation with the agonist, arginine-vasopressin. Herein, we show that the V2R is ubiquitinated following agonist stimulation. V2R-ubiquitination is not observed in a beta-arrestin1,2 deleted mouse fibroblast cell line and is restored following introduction of beta-arrestin2, thus indicating that beta-arrestin2 is required for the ubiquitination of V2R.

Multifaceted roles of beta-arrestins in the regulation of seven-membrane-spanning receptor trafficking and signalling.

Beta-arrestins are cytosolic proteins that bind to activated and phosphorylated G-protein-coupled receptors [7MSRs (seven-membrane-spanning receptors)] and uncouple them from G-protein-mediated second messenger signalling pathways. The binding of beta-arrestins to 7MSRs also leads to new signals via activation of MAPKs (mitogen-activated protein kinases) such as JNK3 (c-Jun N-terminal kinase 3), ERK1/2 (extracellular-signal-regulated kinase 1/2) and p38 MAPKs. By binding to endocytic proteins [clathrin, AP2 (adapter protein 2), NSF (N -ethylmaleimide-sensitive fusion protein) and ARF6 (ADP-ribosylation factor 6)], beta-arrestins also serve as adapters to link the receptors to the cellular trafficking machinery.

Beta-arrestin 2 mediates endocytosis of type III TGF-beta receptor and down-regulation of its signaling.

beta-Arrestins bind to activated seven transmembrane-spanning (7TMS) receptors (G protein-coupled receptors) after the receptors are phosphorylated by G protein-coupled receptor kinases (GRKs), thereby regulating their signaling and internalization. Here, we demonstrate an unexpected and analogous role of beta-arrestin 2 (betaarr2) for the single transmembrane-spanning type III transforming growth factor-beta (TGF-beta) receptor (TbetaRIII, also referred to as betaglycan). Binding of betaarr2 to TbetaRIII was also triggered by phosphorylation of the receptor on its cytoplasmic domain (likely at threonine 841).

Dishevelled 2 recruits beta-arrestin 2 to mediate Wnt5A-stimulated endocytosis of Frizzled 4.

Wnt proteins, regulators of development in many organisms, bind to seven transmembrane-spanning (7TMS) receptors called frizzleds, thereby recruiting the cytoplasmic molecule dishevelled (Dvl) to the plasma membrane.Frizzled-mediated endocytosis of Wg (a Drosophila Wnt protein) and lysosomal degradation may regulate the formation of morphogen gradients. Endocytosis of Frizzled 4 (Fz4) in human embryonic kidney 293 cells was dependent on added Wnt5A protein and was accomplished by the multifunctional adaptor protein beta-arrestin 2 (betaarr2), which was recruited to Fz4 by binding to phosphorylated Dvl2.

Independent beta-arrestin 2 and G protein-mediated pathways for angiotensin II activation of extracellular signal-regulated kinases 1 and 2.

Stimulation of a mutant angiotensin type 1A receptor (DRY/AAY) with angiotensin II (Ang II) or of a wild-type receptor with an Ang II analog ([sarcosine1,Ile4,Ile8]Ang II) fails to activate classical heterotrimeric G protein signaling but does lead to recruitment of beta-arrestin 2-GFP and activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) (maximum stimulation approximately 50% of wild type). This G protein-independent activation of mitogen-activated protein kinase is abolished by depletion of cellular beta-arrestin 2 but is unaffected by the PKC inhibitor Ro-31-8425. In parallel, stimulation of the wild-type angiotensin type 1A receptor with Ang II robustly stimulates ERK1/2 activation with approximately 60% of the response blocked by the PKC inhibitor (G protein dependent) and the rest of the response blocked by depletion of cellular beta-arrestin 2 by small interfering RNA (beta-arrestin dependent).

N-formyl peptide receptors internalize but do not recycle in the absence of arrestins.

Arrestins mediate phosphorylation-dependent desensitization, internalization, and initiation of signaling cascades for the majority of G protein-coupled receptors (GPCRs). Many GPCRs undergo agonist-mediated internalization through arrestin-dependent mechanisms, wherein arrestin serves as an adapter between the receptor and endocytic proteins. To understand the role of arrestins in N-formyl peptide receptor (FPR) trafficking, we stably expressed the FPR in a mouse embryonic fibroblast cell line (MEF) that lacked endogenous arrestin 2 and arrestin 3 (arrestin-deficient).

Beta-arrestin-2 regulates the development of allergic asthma.

Asthma is a chronic inflammatory disorder of the airways that is coordinated by Th2 cells in both human asthmatics and animal models of allergic asthma. Migration of Th2 cells to the lung is key to their inflammatory function and is regulated in large part by chemokine receptors, members of the seven-membrane-spanning receptor family. It has been reported recently that T cells lacking beta-arrestin-2, a G protein-coupled receptor regulatory protein, demonstrate impaired migration in vitro.

Protein kinase A and G protein-coupled receptor kinase phosphorylation mediates beta-1 adrenergic receptor endocytosis through different pathways.

Agonist-induced phosphorylation of beta-adrenergic receptors (beta ARs) by G protein-coupled receptor kinases (GRKs) results in their desensitization followed by internalization. Whether protein kinase A (PKA)-mediated phosphorylation of beta ARs, particularly the beta 1AR subtype, can also trigger internalization is currently not known. To test this, we cloned the mouse wild type beta 1AR (WT beta 1AR) and created 3 mutants lacking, respectively: the putative PKA phosphorylation sites (PKA-beta 1AR), the putative GRK phosphorylation sites (GRK-beta 1AR), and both sets of phosphorylation sites (PKA-/GRK-beta 1AR).

Breath-hold magnetic resonance cholangiopancreatography in the evaluation of malignant pancreaticobiliary obstruction.

Objective: This study was designed to determine the effectiveness of magnetic resonance cholangiopancreatography (MRCP) using a breath-hold single-shot fast spin echo (SSFSE) technique in imaging patients with malignant biliary and/or pancreatic duct obstruction.

Methods: One hundred thirty-one breath-hold MRCP studies in patients with malignant pancreatic and/or biliary obstruction were evaluated. Pathologic diagnoses included pancreatic cancer, biliary malignancy, gallbladder carcinoma, hepatic neoplasms, malignant lymphadenopathy, and ampullary carcinoma.

Purification, crystallization and preliminary X-ray diffraction studies of a complex between G protein-coupled receptor kinase 2 and Gbeta1gamma2.

G protein-coupled receptor kinase 2 (GRK2) phosphorylates activated G protein-coupled receptors (GPCRs), which ultimately leads to their desensitization and/or downregulation. The enzyme is recruited to the plasma membrane via the interaction of its carboxyl-terminal pleckstrin-homology (PH) domain with the beta and gamma subunits of heterotrimeric G proteins (Gbetagamma). An improved purification scheme for GRK2 has been developed, conditions under which GRK2 forms a complex with Gbeta(1)gamma(2) have been determined and the complex has been crystallized in CHAPS detergent micelles.

Keeping G proteins at bay: a complex between G protein-coupled receptor kinase 2 and Gbetagamma.

The phosphorylation of heptahelical receptors by heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor kinases (GRKs) is a universal regulatory mechanism that leads to desensitization of G protein signaling and to the activation of alternative signaling pathways. We determined the crystallographic structure of bovine GRK2 in complex with G protein beta1gamma2 subunits. Our results show how the three domains of GRK2-the RGS (regulator of G protein signaling) homology, protein kinase, and pleckstrin homology domains-integrate their respective activities and recruit the enzyme to the cell membrane in an orientation that not only facilitates receptor phosphorylation, but also allows for the simultaneous inhibition of signaling by Galpha and Gbetagamma subunits.

GIPC interacts with the beta1-adrenergic receptor and regulates beta1-adrenergic receptor-mediated ERK activation.

Beta1-adrenergic receptors, expressed at high levels in the human heart, have a carboxyl-terminal ESKV motif that can directly interact with PDZ domain-containing proteins. Using the beta1-adrenergic receptor carboxyl terminus as bait, we identified the novel beta1-adrenergic receptor-binding partner GIPC in a yeast two-hybrid screen of a human heart cDNA library. Here we demonstrate that the PDZ domain-containing protein, GIPC, co-immunoprecipitates with the beta1-adrenergic receptor in COS-7 cells.

Dopaminergic supersensitivity in G protein-coupled receptor kinase 6-deficient mice.

Brain dopaminergic transmission is a critical component in numerous vital functions, and its dysfunction is involved in several disorders, including addiction and Parkinson's disease. Responses to dopamine are mediated via G protein-coupled dopamine receptors (D1-D5). Desensitization of G protein-coupled receptors is mediated via phosphorylation by members of the family of G protein-coupled receptor kinases (GRK1-GRK7).

G-protein-coupled receptor (GPCR) kinase phosphorylation and beta-arrestin recruitment regulate the constitutive signaling activity of the human cytomegalovirus US28 GPCR.

Phosphorylation of G-protein-coupled receptors (GPCRs) by GRKs and subsequent recruitment of beta-arrestins to agonist-occupied receptors serves to terminate or attenuate signaling by blocking G-proteins from further interaction with the receptors. Human cytomegalovirus encodes a GPCR termed US28 that is homologous to the human chemokine family of GPCRs but differs from the cellular receptors in that it maintains high constitutive activity in the absence of agonist. Although US28 is constitutively active, mechanisms that regulate this activity are unknown.

Routine editing of trainee-generated radiology reports: effect on style quality.

Rationale And Objectives: The authors performed this study to determine the effect of routine editing on the style quality of trainee-generated radiology reports.

Materials And Methods: Trainee-generated reports of 50 body computed tomographic scans obtained at a tertiary care cancer center were edited in a routine fashion by one of two attending radiologists. Three physicians and four radiologists each independently evaluated the randomized unedited and edited reports (n = 100) and rated each report for clarity, brevity, readability, and quality of the impression by using a five-point scale.

Desensitization, internalization, and signaling functions of beta-arrestins demonstrated by RNA interference.

Beta-arrestins bind to activated G protein-coupled receptor kinase-phosphorylated receptors, which leads to their desensitization with respect to G proteins, internalization via clathrin-coated pits, and signaling via a growing list of "scaffolded" pathways. To facilitate the discovery of novel adaptor and signaling roles of beta-arrestins, we have developed and validated a generally applicable interfering RNA approach for selectively suppressing beta-arrestins 1 or 2 expression by up to 95%. Beta-arrestin depletion in HEK293 cells leads to enhanced cAMP generation in response to beta(2)-adrenergic receptor stimulation, markedly reduced beta(2)-adrenergic receptor and angiotensin II receptor internalization and impaired activation of the MAP kinases ERK 1 and 2 by angiotensin II.

Trafficking patterns of beta-arrestin and G protein-coupled receptors determined by the kinetics of beta-arrestin deubiquitination.

Agonist-dependent internalization of G protein-coupled receptors via clathrin-coated pits is dependent on the adaptor protein beta-arrestin, which interacts with elements of the endocytic machinery such as AP2 and clathrin. For the beta(2)-adrenergic receptor (beta(2)AR) this requires ubiquitination of beta-arrestin by E3 ubiquitin ligase, Mdm2. Based on trafficking patterns and affinity of beta-arrestin, G protein-coupled receptors are categorized into two classes.

beta-Arrestin-mediated PDE4 cAMP phosphodiesterase recruitment regulates beta-adrenoceptor switching from Gs to Gi.

Phosphorylation of the beta(2) adrenoreceptor (beta(2)AR) by cAMP-activated protein kinase A (PKA) switches its predominant coupling from stimulatory guanine nucleotide regulatory protein (G(s)) to inhibitory guanine nucleotide regulatory protein (G(i)). beta-Arrestins recruit the cAMP-degrading PDE4 phosphodiesterases to the beta(2)AR, thus controlling PKA activity at the membrane. Here we investigate a role for PDE4 recruitment in regulating G protein switching by the beta(2)AR.