Showing papers by "Robert J. Lefkowitz published in 2003"

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

Abstract: 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 β-arrestin 2-GFP and activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) (maximum stimulation ≈50% of wild type). This G protein-independent activation of mitogen-activated protein kinase is abolished by depletion of cellular β-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 ≈60% of the response blocked by the PKC inhibitor (G protein dependent) and the rest of the response blocked by depletion of cellular β-arrestin 2 by small interfering RNA (β-arrestin dependent). These findings imply the existence of independent G protein- and β-arrestin 2-mediated pathways leading to ERK1/2 activation and the existence of distinct “active” conformations of a seven-membrane-spanning receptor coupled to each.

Regulation of G Protein-Coupled Receptor Kinases and Arrestins During Receptor Desensitization

Abstract: With at least 1000 family members encoded by mammalian genomes, the G protein-coupled receptors (GPCRs) represent the most diverse group of signaling proteins known ([Bockaert and Pin, 1999][1]). GPCRs are involved in the regulation of a wide variety of physiological processes including, but not

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

Abstract: 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. Agonist-promoted ubiquitination of beta-arrestins is a prerequisite for their role in receptor internalization, as well as a determinant of the differing trafficking patterns of distinct classes of receptors. Recently, beta-arrestins have also been implicated as playing novel roles in cellular chemotaxis and apoptosis. By virtue of their ability to bind, in a stimulus-dependent fashion, to 7MSRs as well as to different classes of cellular proteins, beta-arrestins serve as versatile adapter proteins that regulate the signalling and trafficking of the receptors.

Keeping G Proteins at Bay: A Complex Between G Protein-Coupled Receptor Kinase 2 and Gßγ

Abstract: 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.

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

Abstract: Phosphorylation of the β2 adrenoreceptor (β2AR) by cAMP-activated protein kinase A (PKA) switches its predominant coupling from stimulatory guanine nucleotide regulatory protein (Gs) to inhibitory guanine nucleotide regulatory protein (Gi). β-Arrestins recruit the cAMP-degrading PDE4 phosphodiesterases to the β2AR, thus controlling PKA activity at the membrane. Here we investigate a role for PDE4 recruitment in regulating G protein switching by the β2AR. In human embryonic kidney 293 cells overexpressing a recombinant β2AR, stimulation with isoprenaline recruits β-arrestins 1 and 2 as well as both PDE4D3 and PDE4D5 to the receptor and stimulates receptor phosphorylation by PKA. The PKA phosphorylation status of the β2AR is enhanced markedly when cells are treated with the selective PDE4-inhibitor rolipram or when they are transfected with a catalytically inactive PDE4D mutant (PDE4D5-D556A) that competitively inhibits isoprenaline-stimulated recruitment of native PDE4 to the β2AR. Rolipram and PDE4D5-D556A also enhance β2AR-mediated activation of extracellular signal-regulated kinases ERK1/2. This is consistent with a switch in coupling of the receptor from Gs to Gi, because the ERK1/2 activation is sensitive to both inhibitors of PKA (H89) and Gi (pertussis toxin). In cardiac myocytes, the β2AR also switches from Gs to Gi coupling. Treating primary cardiac myocytes with isoprenaline induces recruitment of PDE4D3 and PDE4D5 to membranes and activates ERK1/2. Rolipram robustly enhances this activation in a manner sensitive to both pertussis toxin and H89. Adenovirus-mediated expression of PDE4D5-D556A also potentiates ERK1/2 activation. Thus, receptor-stimulated β-arrestin-mediated recruitment of PDE4 plays a central role in the regulation of G protein switching by the β2AR in a physiological system, the cardiac myocyte.

Dishevelled 2 Recruits ß-Arrestin 2 to Mediate Wnt5A-Stimulated Endocytosis of Frizzled 4

Abstract: 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 membraneFrizzled-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 These findings provide a previously unrecognized mechanism for receptor recruitment of beta-arrestin and demonstrate that Dvl plays an important role in the endocytosis of frizzled, as well as in promoting signaling

ß-Arrestin 2 Mediates Endocytosis of Type III TGF-ß Receptor and Down-Regulation of Its Signaling

Abstract: β-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 β-arrestin 2 (βarr2) for the single transmembrane–spanning type III transforming growth factor–β (TGF-β) receptor (TβRIII, also referred to as betaglycan). Binding of βarr2 to TβRIII was also triggered by phosphorylation of the receptor on its cytoplasmic domain (likely at threonine 841). However, such phosphorylation was mediated by the type II TGF-β receptor (TβRII), which is itself a kinase, rather than by a GRK. Association with βarr2 led to internalization of both receptors and down-regulation of TGF-β signaling. Thus, the regulatory actions of β-arrestins are broader than previously appreciated, extending to the TGF-β receptor family as well.

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

Abstract: β-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 β-arrestins, we have developed and validated a generally applicable interfering RNA approach for selectively suppressing β-arrestins 1 or 2 expression by up to 95%. β-Arrestin depletion in HEK293 cells leads to enhanced cAMP generation in response to β2-adrenergic receptor stimulation, markedly reduced β2-adrenergic receptor and angiotensin II receptor internalization and impaired activation of the MAP kinases ERK 1 and 2 by angiotensin II. This approach should allow discovery of novel signaling and regulatory roles for the β-arrestins in many seven-membrane-spanning receptor systems.

β-Arrestin-2 regulates the development of allergic asthma

Abstract: 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 β-arrestin-2, a G protein‐coupled receptor regulatory protein, demonstrate impaired migration in vitro. Here we show that allergen-sensitized mice having a targeted deletion of the β-arrestin-2 gene do not accumulate T lymphocytes in their airways, nor do they demonstrate other physiological and inflammatory features characteristic of asthma. In contrast, the airway inflammatory response to LPS, an event not coordinated by Th2 cells, is fully functional in mice lacking β-arrestin-2. β-arrestin-2‐deficient mice demonstrate OVA-specific IgE responses, but have defective macrophage-derived chemokine‐mediated CD4 + T cell migration to the lung. This report provides the first evidence that β-arrestin-2 is required for the manifestation of allergic asthma. Because β-arrestin2 regulates the development of allergic inflammation at a proximal step in the inflammatory cascade, novel therapies focused on this protein may prove useful in the treatment of asthma.

Increased Acute Inflammation, Leukotriene B4-Induced Chemotaxis, and Signaling in Mice Deficient for G Protein-Coupled Receptor Kinase 6

Abstract: Directed migration of polymorphonuclear neutrophils (PMN) is required for adequate host defense against invading organisms and leukotriene B4 (LTB4) is one of the most potent PMN chemoattractants. LTB4 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. Recently, it has been shown that the human BLT1 is a substrate for GRK6. To investigate the physiological importance of GRK6 for inflammation and LTB4 signaling in PMN, we used GRK6-deficient mice. The acute inflammatory response (ear swelling and influx of PMN into the ear) after topical application of arachidonic acid was significantly increased in GRK6−/− mice. In vitro, GRK6−/− PMN showed increased chemokinetic and chemotactic responses to LTB4. GRK6−/− PMN respond to LTB4 with a prolonged increase in intracellular calcium and prolonged actin polymerization, suggesting impaired LTB4 receptor desensitization in the absence of GRK6. However, pre-exposure to LTB4 renders both GRK6−/− as well as wild-type PMN refractory to restimulation with LTB4, indicating that the presence of GRK6 is not required for this process to occur. In conclusion, GRK6 deficiency leads to prolonged BLT1 signaling and increased neutrophil migration.

Purification, crystallization and preliminary X‐ray diffraction studies of a complex between G protein‐coupled receptor kinase 2 and Gβ1γ2

Abstract: 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. Crystals of the GRK2-Gbetagamma complex belong to space group C2 and have unit-cell parameters a = 187.0, b = 72.1, c = 122.0 A, beta = 115.2 degrees. A complete data set has been collected to 3.2 A resolution with Cu Kalpha radiation.

Methods of screening compounds for grk6 modulating activity

Abstract: The present invention relates to methods of treating disease by altering G protein coupled receptor kinase (GRK) 6. This may be done by altering the expression or activity of the protein, for example. The present invention may be used for disease diagnosis, by detecting the expression or activity of GRK6. The present invention relates to a GRK6 deficient mouse, GRK6 splice variants, and methods of use. The present invention also relates to methods of identifying compounds that alter GRK6 activity. The present invention relates to disease treatment by altering GRK6 expression or activity.

A Magnificent Time With the “Magnificent Seven” Transmembrane Spanning Receptors

Abstract: The largest, most ubiquitous, and most versatile of the receptor gene families is that which encodes the seven transmembrane spanning receptors. In the cardiovascular system, such receptors regulate, for example, the rate and force of cardiac contraction, peripheral arterial resistance, and various aspects of renal function. The larger superfamily of receptors also regulates everything from sensory perception (vision, smell, taste) to hormonal and neurotransmitter signaling, to immune functions such as chemotaxis. There is virtually no area of human physiology in which they are not implicated. A majority of prescription drugs sold target such receptors either directly or indirectly. In the field of cardiovascular medicine, this is exemplified by α- and β-adrenergic receptor agonists and antagonists, angiotensin receptor blockers, and angiotensin-converting enzyme inhibitors. A profound alteration in our understanding of these receptors occurred during the 1970s and 1980s, which transformed everything from the way they are viewed, to how they are studied, to how new drugs are discovered. I provide here a very personal recollection and perspective of my own voyage of discovery during this time, of its scientific antecedents, and of the technical and conceptual barriers that needed to be scaled. The notion that biologically active substances initiate their actions by binding with high affinity and selectivity to some “receptive substance” on cells dates back about a hundred years. Early work of Ehrlich on interactions of antigens with cells and of chemotherapeutic agents with their targets was made even more explicit by J.N. Langley and H.H. Dale who, during the earliest years of the 20th century, studied actions of cholinergic and adrenergic agonists and antagonists on various target organs. Based on this early work, the period from about 1920 to 1970 was characterized by the development of classical theories of receptor action based on the law of mass action. This …