Showing papers by "Robert J. Lefkowitz published in 1995"
TL;DR: Transgenic mice created with cardiac-specific overexpression of the beta-adrenergic receptor kinase-1 (beta ARK1) or a beta ARK inhibitor displayed enhanced cardiac contractility in vivo with or without isoproterenol, demonstrating the important role of Beta ARK in modulating in vivo myocardial function.
Abstract: Transgenic mice were created with cardiac-specific overexpression of the beta-adrenergic receptor kinase-1 (beta ARK1) or a beta ARK inhibitor. Animals overexpressing beta ARK1 demonstrated attenuation of isoproterenol-stimulated left ventricular contractility in vivo, dampening of myocardial adenylyl cyclase activity, and reduced functional coupling of beta-adrenergic receptors. Conversely, mice expressing the beta ARK inhibitor displayed enhanced cardiac contractility in vivo with or without isoproterenol. These animals demonstrate the important role of beta ARK in modulating in vivo myocardial function. Because increased amounts of beta ARK1 and diminished cardiac beta-adrenergic responsiveness characterize heart failure, these animals may provide experimental models to study the role of beta ARK in heart disease.
711 citations
Journal Article•
710 citations
TL;DR: It is demonstrated that activation of MAP kinase by Gi-coupled receptors is preceded by the Gβγ-mediated tyrosine phosphorylation of She, leading to an increased functional association between She, Grb2 and Sos.
Abstract: Mitogen-activated protein (MAP) kinases mediate the phosphorylation and activation of nuclear transcription factors that regulate cell growth. MAP kinase activation may result from stimulation of either tyrosine-kinase (RTK) receptors, which possess intrinsic tyrosine kinase activity, or G-protein-coupled receptors (GPCR). RTK-mediated mitogenic signalling involves a series of SH2- and SH3-dependent protein-protein interactions between tyrosine-phosphorylated receptor, Shc, Grb2 and Sos, resulting in Ras-dependent MAP kinase activation. The beta gamma subunits of heterotrimeric G proteins (G beta gamma) also mediate Ras-dependent MAP kinase activation by an as-yet unknown mechanism. Here we demonstrate that activation of MAP kinase by Gi-coupled receptors is preceded by the G beta gamma-mediated tyrosine phosphorylation of Shc, leading to an increased functional association between Shc, Grb2 and Sos. Moreover, disruption of the Shc-Grb2-Sos complex blocks G beta gamma-mediated MAP kinase activation, indicating that G beta gamma does not mediate MAP kinase activation by a direct interaction with Sos. These results indicate that G beta gamma-mediated MAP kinase activation is initiated by a tyrosine phosphorylation event and proceeds by a pathway common to both GPCRs and RTKs.
532 citations
TL;DR: All GRKs appear to play the same general cellular role of desensitizing activated G protein‐coupled receptors, but utilize distinctly individual means to the same end, which are just beginning to be defined.
Abstract: G protein-coupled receptor kinases (GRKs) are a family of serine/threonine protein kinases that specifically recognize agonist-occupied, activated G protein-coupled receptor proteins as substrates. Phosphorylation of an activated receptor by a GRK terminates signaling by that receptor, by initiating the uncoupling of the receptor from heterotrimeric G proteins. Six distinct mammalian GRKs are known, which differ in tissue distribution and in regulatory properties. The intracellular localization of GRKs to membrane-bound receptor substrates is the most important known regulatory feature of these enzymes. Rhodopsin kinase (GRK1) requires a post-translationally added farnesyl isoprenoid to bind to light-activated rhodopsin. The beta-adrenergic receptor kinases (GRK2 and GRK3) associate with heterotrimeric G protein beta gamma-subunits, released upon receptor activation of G proteins, for membrane anchorage. The recently-described GRKs 4, 5, and 6 comprise a distinct subgroup of GRKs. These kinases utilize di...
523 citations
TL;DR: The role of Gβ in Gi- and Gq-coupled receptor-mediated PI hydrolysis and MAPK activation is defined and direct stimulation with Gβ resulted in MAPKactivation that was sensitive to inhibition by expression of βARKct, RasN17, or NΔRaf or by PTK inhibitors, but insensitive to PKC depletion.
442 citations
TL;DR: It is reported that the binding of G and lipid to the PH domain of the β-adrenergic receptor kinase (βARK) synergistically enhances agonist-dependent receptor phosphorylation and that both PH domain-binding ligands are required for membrane association of the kinase.
371 citations
TL;DR: It is concluded that rapid agonist-induced desensitization of the beta 1AR involves phosphorylation of the receptor by both PKA and at least beta ARK1 in intact cells.
323 citations
TL;DR: The specific and Ca-dependent Rv/RK interaction is necessary for the inhibitory effect of Rv on rhodopsin phosphorylation and may play an important role in photoreceptor light adaptation.
278 citations
TL;DR: The region defined by residues 956 to 982 of adenylyl cyclase 2 may contain determinants important for receiving signals from G beta gamma, which is conserved in regions of potassium channels and beta-adrenergic receptor kinases that participate in G beta Gamma interactions.
Abstract: Receptor-mediated activation of heterotrimeric guanine nucleotide-binding proteins (G proteins) results in the dissociation of alpha from beta gamma subunits, thereby allowing both to regulate effectors. Little is known about the regions of effectors required for recognition of G beta gamma. A peptide encoding residues 956 to 982 of adenylyl cyclase 2 specifically blocked G beta gamma stimulation of adenylyl cyclase 2, phospholipase C-beta 3, potassium channels, and beta-adrenergic receptor kinase as well as inhibition of calmodulin-stimulated adenylyl cyclases, but had no effect on interactions between G beta gamma and G alpha o. Substitutions in this peptide identified a functionally important motif, Gln-X-X-Glu-Arg, that is also conserved in regions of potassium channels and beta-adrenergic receptor kinases that participate in G beta gamma interactions. Thus, the region defined by residues 956 to 982 of adenylyl cyclase 2 may contain determinants important for receiving signals from G beta gamma.
277 citations
Journal Article•
TL;DR: It is concluded that short term desensitization of the delta OR involves phosphorylation of the receptor by one or more G protein-coupled receptor kinases.
Abstract: With chronic opiate use, opioid receptor desensitization may be one of the important mechanisms underlying the development of opiate tolerance and addiction. Opioid receptors belong to the G protein-coupled receptor superfamily. In this study, the mouse delta-opioid receptor (delta OR) was used in a model system to investigate the role of opioid receptor phosphorylation in receptor desensitization. When expressed in 293 cells and exposed to agonist, the delta OR underwent receptor-specific desensitization within 10 min. This agonist-induced desensitization corresponded temporally to a 3-fold increase in receptor phosphorylation. Phorbol ester, but not forskolin, also stimulated phosphorylation of the delta OR in 293 cells. Although down-regulation of protein kinase C failed to affect agonist-induced receptor phosphorylation, it abolished phorbol ester-induced receptor phosphorylation. Agonist-induced delta OR phosphorylation must therefore involve kinases other than protein kinase C. Whereas overexpression of a dominant negative mutant (K220R) of beta-adrenergic receptor kinase-1 (beta ARK1) in 293 cells significantly reduced agonist-dependent phosphorylation of the delta OR, overexpression of beta ARK1 or G protein-coupled receptor kinase-5 significantly enhanced this phosphorylation. Concordantly, beta ARK1-K220R overexpression reduced agonist-dependent delta OR desensitization, whereas beta ARK1 overexpression enhanced this densensitization. We conclude that short term desensitization of the delta OR involves phosphorylation of the receptor by one or more G protein-coupled receptor kinases.
228 citations
TL;DR: In Rat 1 fibroblasts, stimulation of mitogen-activated protein (MAP) kinase via the IGF1 receptor and the Gi-coupled receptor for lysophosphatidic acid (LPA), but not via the EGF receptor, is sensitive both to pertussis toxin treatment and to cellular expression of a specific Gβγ subunit-binding peptide.
TL;DR: The subcellular distribution and substrate specificity of the GRP suggests significant differences between it and previously characterized forms of PP-2A, which has previously been characterized as a soluble enzyme, yet negligible soluble GRP activity was observed.
Abstract: Phosphorylation of G-protein-coupled receptors plays an important role in regulating their function. In this study the G-protein-coupled receptor phosphatase (GRP) capable of dephosphorylating G-protein-coupled receptor kinase-phosphorylated receptors is described. The GRP activity of bovine brain is a latent oligomeric form of protein phosphatase type 2A (PP-2A) exclusively associated with the particulate fraction. GRP activity is observed only when assayed in the presence of protamine or when phosphatase-containing fractions are subjected to freeze/thaw treatment under reducing conditions. Consistent with its identification as a member of the PP-2A family, the GRP is potently inhibited by okadaic acid but not by I-2, the specific inhibitor of protein phosphatase type 1. Solubilization of the membrane-associated GRP followed by gel filtration in the absence of detergent yields a 150-kDa peak of latent receptor phosphatase activity. Western blot analysis of this phosphatase reveals a likely subunit composition of AB alpha C. PP-2A of this subunit composition has previously been characterized as a soluble enzyme, yet negligible soluble GRP activity was observed. The subcellular distribution and substrate specificity of the GRP suggests significant differences between it and previously characterized forms of PP-2A.
TL;DR: The βγ-subunit complex of G proteins mediates many of the functions associated with G-protein-coupled receptor signaling and may even provide a means to link G proteins to RTK-initiated cascades.
TL;DR: Results suggest that G beta gamma-stimulated Shc phosphorylation represents an early step in the pathway leading to p21ras activation, similar to the mechanism utilized by growth factor tyrosine kinase receptors.
Abstract: The mechanism of mitogen-activated protein (MAP) kinase activation by pertussis toxin-sensitive Gi-coupled receptors is known to involve the beta gamma subunits of heterotrimeric G proteins (G beta gamma), p21ras activation, and an as-yet-unidentified tyrosine kinase. To investigate the mechanism of G beta gamma-stimulated p21ras activation, G beta gamma-mediated tyrosine phosphorylation was examined by overexpressing G beta gamma or alpha 2-C10 adrenergic receptors (ARs) that couple to Gi in COS-7 cells. Immunoprecipitation of phosphotyrosine-containing proteins revealed a 2- to 3-fold increase in the phosphorylation of two proteins of approximately 50 kDa (designated as p52) in G beta gamma-transfected cells or in alpha 2-C10 AR-transfected cells stimulated with the agonist UK-14304. The latter response was pertussis toxin sensitive. These proteins (p52) were also specifically immunoprecipitated with anti-Shc antibodies and comigrated with two Shc proteins, 46 and 52 kDa. The G beta gamma- or alpha 2-C10 AR-stimulated p52 (Shc) phosphorylation was inhibited by coexpression of the carboxyl terminus of beta-adrenergic receptor kinase (a G beta gamma-binding pleckstrin homology domain peptide) or by the tyrosine kinase inhibitors genistein and herbimycin A, but not by a dominant negative mutant of p21ras. Worthmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K) inhibited phosphorylation of p52 (Shc), implying involvement of PI3K. These results suggest that G beta gamma-stimulated Shc phosphorylation represents an early step in the pathway leading to p21ras activation, similar to the mechanism utilized by growth factor tyrosine kinase receptors.
TL;DR: The results suggest that the last PH subdomain and its neighboring sequences within the carboxyl terminus of βARK, including Trp643, Leu647, and residues Lys663-Arg669, are critical for Gβγ binding while Tr p643 and residues Asp635-Glu639 are important for the PH domain to form the correct structure for binding to PIP2.
TL;DR: It is plausible that GRK3 and β -arrestin2 function in sperm to regulate putative chemoreceptor responses and consistent with a role for these proteins in transducing chemotactic signals.
Abstract: The identification of transcripts encoding putative olfactory receptors in mammalian germ cells (1) has generated the hypothesis that olfactory receptors may serve a chemosensory role in sperm Chemotaxis during fertilization. We have sought to identify and localize these receptors and their regulatory machinery in rat sperm in order to gain further insight into mammalian sperm Chemotaxis and odorant receptor physiology. We conducted reverse transcription-polymerase chain reaction (RT-PCR) using degenerate primers directed against sequences conserved across members of the known odorant receptor family to identify transcripts from testis and round spermatids. Western analysis and immunohistochemistry were performed using antibodies raised against two peptide sequences conserved among odorant receptors and using fusion protein antibodies to G-protein receptor kinase 3 (GRK3/βARK2) and β-arrestin2. We detected transcripts encoding putative odorant receptors in both testis and round spermatids of the adult rat. Restriction digests of the PCR products demonstrated the existence of multiple gene products. Two anti-odorant receptor antibodies specifically recognized a 64 kD band in rat sperm preparations by Western blot. The proteins GRK3 and β-arrestin2, implicated in olfactory desensitization, were detected in sperm cytosolic extracts using Western analysis. Immunohistochemistry colocalized putative odorant receptors, GRK3 and β-arrestin2 to elongating spermatids in the testis and to the midpiece of mature sperm.
TL;DR: The data suggest that the PH domain peptides behave as specific antagonists of Gβγ-mediated signaling in intact cells and that interactions between PH domains and G βγ subunits or structurally related proteins may play a role in the activation of mitogenic signaling pathways by G protein-coupled receptors.
TL;DR: G beta gamma inhibitors derived from the beta-adrenergic kinase 1 (beta ARK1) were used to assess the relative importance of G beta gamma in IK.ACh activation and provide further evidence for the role of Gbeta gamma in the activation of IK .
Abstract: The cardiac muscarinic potassium channel (IK.ACh) is activated by a G protein upon receptor stimulation with acetylcholine. The G protein subunit responsible for activation (G alpha versus G beta gamma) has been disputed. We used G beta gamma inhibitors derived from the beta-adrenergic kinase 1 (beta ARK1) to assess the relative importance of G beta gamma in IK.ACh activation. In rabbit atrial myocytes, IK.ACh had a conductance of 49 +/- 6.2 pS. In inside-out patches, the mean open time was 1.60 +/- 0.57 ms, mean time constant (tau o) was 1.59 +/- 0.53 ms, and mean closed time was 3.02 +/- 1.35 ms (n = 38). beta ARK1 is a G beta gamma-sensitive enzyme that interacts with G beta gamma through a defined sequence near its carboxyl terminus. A 28-amino-acid peptide derived from the carboxyl terminus of beta ARK1 (peptide G) increased the closed time to 10.04 ms (P < .001) and decreased opening probability (NPo) by 71% (P < .001). Fusion proteins containing the entire carboxyl terminus of beta ARK1, glutathione S-transferase beta ARK1ct and hexahistidine beta ARK1ct, decreased NPo by 67% (P = .03) and 48% (P = .009), respectively. They also both significantly increased the closed time. None of the inhibitors affected mean open time or channel amplitude. A control peptide derived from a neighboring region of beta ARK1 had no significant effect on IK.ACh activity. These results provide further evidence for the role of G beta gamma in the activation of IK.ACh.
TL;DR: The award of the 1994 Nobel Prize in Physiology or Medicine to Alfred G. Gilman and Martin Rodbell for the discovery of G (guanine nucleotide–binding) proteins and their role in cellular signal tra...
Abstract: The award of the 1994 Nobel Prize in Physiology or Medicine to Alfred G. Gilman and Martin Rodbell for the discovery of G (guanine nucleotide–binding) proteins and their role in cellular signal tra...
TL;DR: Transgenic mice with intense cardiac expression of a human beta-adrenergic receptor gene were engineered and shown to display marked improvements in baseline myocardial and left ventricular function, suggesting a novel approach for increasing myocardIAL function with important clinical implications.
TL;DR: An efficient method for beta 2-AR purification was developed using a recombinant receptor with an eight amino acid epitope at its C-terminus, which is recognized by KT3-monoclonal antibody.
01 Jan 1995
TL;DR: The chapter discusses the human b-adrenergic receptor kinases (bARK) along with its subunit and domain structure and the amino acid sequence of bARK, which leads to homologous, or agonist-specific, desensitization.
Abstract: The chapter discusses the human b-adrenergic receptor kinases (bARK) along with its subunit and domain structure and the amino acid sequence of bARK. Desensitization is mediated in part by bARK-mediated phosphorylation of the receptor. b-Adrenergic receptor kinases, two isoforms of which are currently known (bARK-1 and -2), phosphorylate the agonist-occupied forms of several G protein-coupled receptors. Phosphorylation is thought to be followed by the binding of another protein, b-arrestin, which diminishes the functional coupling of the receptor to the G protein. This mechanism, thereby leads to homologous, or agonist-specific, desensitization. bARK activity is normally assayed as the agonist-dependent transfer of phosphate from [g32 P]ATP to purified, reconstituted b2AR. However, the light-dependent transfer of phosphate to urea-washed, rod outer segment membranes can also be utilized as an assay.
01 Jan 1995
TL;DR: The localization of RhK mRNA is highly specific to the retina and suggests that rhodopsin is the primary in vivo substrate of the kinase, consistent with other biochemical similarities between the mammalian pineal gland and retina.
Abstract: The chapter focuses on the protein Rhodopsin kinase (RhK) that catalyzes phosphorylation of photolyzed rhodopsin (Rho*), presumably the metarhodopsin II conformer, at multiple serine and threonine residues located exclusively in the C-terminal region. In addition to phosphorylating rhodopsin, RhK phosphorylates in vitro the red pigment, iodopsin. The substrate specificity of RhK appears therefore, not to be dictated by amino acid sequence but by the conformation of photolyzed rhodopsin. RhK preferentially phosphorylates serine and threonine residues located in an acidic environment. RhK is assayed as a light-dependent transfer of radioactivity from [g32P]ATP to urea-washed, rod outer segment membranes. The localization of RhK mRNA is highly specific to the retina. It is also present in lower amounts in the pineal gland, consistent with other biochemical similarities between the mammalian pineal gland and retina. This mRNA specificity suggests that rhodopsin is the primary in vivo substrate of the kinase. Immunocytochemistry has shown that RhK is present in both rods and cones.