Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The beta-adrenergic receptor kinase (beta ARK) is a member of growing family of G protein coupled receptor kinases (GRKs). beta ARK and other members of the GRK family play a role in the mechanism of agonist-specific desensitization by virtue of their ability to phosphorylate G protein-coupled receptors in an agonist-dependent manner. beta ARK activation is known to occur following the interaction of the kinase with the agonist-occupied form of the receptor substrate and heterotrimeric G protein beta gamma subunits. Recently, lipid regulation of GRK2, GRK3, and GRK5 have also been described. Using a mixed micelle assay, GRK2 (beta ARK1) was found to require phospholipid in order to phosphorylate the beta 2-adrenergic receptor. As determined with a nonreceptor peptide substrate of beta ARK, catalytic activity of the kinase increased in the presence of phospholipid without a change in the Km for the peptide. Data obtained with the heterobifunctional cross-linking agent N-3-[125I]iodo-4-azidophenylpropionamido-S-(2-thiopyridyl)-c ysteine ([125I]ACTP) suggests that the activation by phospholipid was associated with a conformational change in the kinase. [125I]ACTP incorporation increased 2-fold in the presence of crude phosphatidylcholine, and this increase in [125I]ACTP labeling is completely blocked by the addition of MgATP. Furthermore, proteolytic mapping was consistent with the modification of a distinct site when GRK2 was labeled in the presence of phospholipid. While an acidic phospholipid specificity was demonstrated using the mixed micelle phosphorylation assay, a notable exception was observed with PIP2. In the presence of PIP2, kinase activity as well as [125I]ACTP labeling was inhibited. These data demonstrate the direct regulation of GRK2 activity by phospholipids and supports the hypothesis that this effect is the result of a conformational change within the kinase.
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PMID:The beta-adrenergic receptor kinase (GRK2) is regulated by phospholipids. 767 71

Guanine nucleotide binding protein (G-protein)-coupled receptor kinases (GRKs) specifically phosphorylate the agonist-occupied form of G-protein-coupled receptors such as the beta 2-adrenergic receptor and rhodopsin. The best characterized members of this family include the beta-adrenergic receptor kinase (beta ARK) and rhodopsin kinase. To identify additional members of the GRK family, the polymerase chain reaction was used to amplify human heart cDNA using degenerate oligonucleotide primers from highly conserved regions unique to the GRK family. Here we report the isolation of a cDNA that encodes a 590-amino acid protein kinase, termed GRK5, which has 34.8% and 47.2% amino acid identities with beta ARK and rhodopsin kinase, respectively. Interestingly, GRK5 has an even higher homology with Drosophila GPRK-2 (71.0% identity) and the recently identified human IT11 (69.1% identity). Northern blot analysis of GRK5 with selected human tissues reveals a message of approximately 3 kilobases with highest levels in heart, placenta, lung > skeletal muscle > brain, liver, pancreas > kidney. GRK5, overexpressed in Sf9 insect cells using the baculovirus system, was able to phosphorylate rhodopsin in a light-dependent manner. In addition, GRK5 neither contains a consensus sequence for isoprenylation like rhodopsin kinase nor is activated by G-protein beta gamma subunits like beta ARK1. Thus, GRK5 represents a member of the GRK family that likely has a unique physiological role.
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PMID:Cloning and expression of GRK5: a member of the G protein-coupled receptor kinase family. 768 6

Two types of proteins play a major role in determining homologous desensitization of G-coupled receptors: beta-adrenergic receptor kinase (beta ARK), which phosphorylates the agonist-occupied receptor and its functional cofactor, beta-arrestin. Both beta ARK and beta-arrestin are members of multigene families. The family of G-protein-coupled receptor kinases includes rhodopsin kinase, beta ARK1, beta ARK2, IT11-A (GRK4), GRK5, and GRK6. The arrestin/beta-arrestin gene family includes arrestin (also known as S-antigen), beta-arrestin 1, and beta-arrestin 2. Here we report the chromosome mapping of the human genes for arrestin (SAG), beta-arrestin 2 (ARRB2), and beta ARK2 (ADRBK2) by fluorescence in situ hybridization (FISH). FISH results confirmed the assignment of the gene coding for arrestin (SAG) to chromosome 2 and allowed us to refine its localization to band q37. The gene coding for beta-arrestin 2 (ARRB2) was mapped to chromosome 17p13 and that coding for beta ARK2 (ADRBK2) to chromosome 22q11.
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PMID:Chromosome mapping of the human arrestin (SAG), beta-arrestin 2 (ARRB2), and beta-adrenergic receptor kinase 2 (ADRBK2) genes. 769 43

Beta-adrenergic receptor kinase (beta ARK) is a serine-threonine kinase involved in the process of homologous desensitization of G-coupled receptors. beta ARK is a member of a multigene family, consisting of six known subtypes, also named G protein-coupled receptor kinases (GRK 1-6). In this study we investigated the expression of GRKs during the process of T cell activation, which is of fundamental importance in regulating immune responses. T cell activation was induced by exposing mononuclear leukocytes (MNL) to PHA and confirmed by tritiated thymidine incorporation measurement. A substantial increase of GRK activity (as measured by in vitro phosphorylation of rhodopsin) was found after 48 h (331 +/- 80% of controls) and 72 h (347 +/- 86% of controls) of exposure to PHA. A threefold increase of beta ARK1 immunoreactivity was found in MNL exposed to PHA for 72 h. Persistent activation of protein kinase C (PKC) by 10 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) was able to increase beta ARK activity to the same extent as PHA, suggesting a PKC-mediated mechanism. The kinetic of beta-adrenergic-stimulated cAMP production was substantially modified in TPA and PHA-activated cells, indicating that the increased GRK activity resulted in an increased beta-adrenergic homologous desensitization. A three- to fourfold increase in GRK activity was also observed in a population of T cell blasts (> 97% CD3+) exposed to PHA for 48-72 h. A significant increase in beta ARK1 and beta ARK2 mRNA expression was observed 48 h after mitogen stimulation, while mRNA expression of GRK5 and GRK6 was not changed. In conclusion our data show that the expression of GRK subtypes is actively and selectively modulated according to the functional state of T lymphocytes.
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PMID:Regulation of G protein-coupled receptor kinase subtypes in activated T lymphocytes. Selective increase of beta-adrenergic receptor kinase 1 and 2. 781 17

Agonist-dependent phosphorylation of G protein-coupled receptors (GPRs) by G protein-coupled receptor kinases (GRKs) is proposed to be a key event initiating homologous receptor desensitization. A technical limitation hindering identification of GPRs as GRK substrates has been the necessity to use purified and reconstituted receptors in GRK assays. Here, the human m2 and human m3 (hm3) muscarinic cholinergic receptors (mAChRs), which couple to attenuation of adenylyl cyclase and stimulation of phospholipase C, respectively, were expressed in Spodoptera frugiperda insect cells and an in vitro approach to studying GPR phosphorylation by GRKs in crude membranes was developed. The m2 mAChR, a known substrate of certain GRKs, was used to validate the approach. The GRK isoform beta-adrenergic receptor kinase (beta ARK)1 phosphorylated the membrane-bound human m2 mAChRs in an agonist-dependent manner. The results demonstrated that endogenous membrane-bound beta gamma subunits of G proteins stimulated the phosphorylation of the membrane-bound m2 mAChR. To reveal new GRK substrates, we tested the expressed hm3 mAChRs. The membrane-bound hm3 mAChRs were phosphorylated by beta ARK1 in an agonist-dependent, G beta gamma-enhanced manner. This is the first demonstration that hm3 mAChRs can serve as substrates for GRKs. The stoichiometry of receptor phosphorylation was approximately 2 mol of phosphate/mol of receptors in the absence of G beta gamma and approximately 4 mol of phosphate/mol of receptors upon addition of G beta gamma. When the specificity of various GRKs towards mAChRs was assessed, beta ARK2 phosphorylated the agonist-activated hm3 mAChRs as efficiently as did beta ARK1; however, neither GRK5 nor GRK6 significantly phosphorylated the hm3 mAChRs under similar conditions. The approach of studying GRK-mediated phosphorylation of GPRs in their membrane-bound state identified the hm3 mAChRs as new substrates for GRKs. This approach should be valuable in identifying other new substrates of GRKs and should aid in studies that elucidate GRK/GPR pairing.
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PMID:Agonist-dependent phosphorylation of human muscarinic receptors in Spodoptera frugiperda insect cell membranes by G protein-coupled receptor kinases. 787 29

G protein-coupled receptor kinases (GRKs), such as rhodopsin kinase and beta-adrenergic receptor kinase (beta ARK), are involved in mediating agonist-specific phosphorylation and desensitization of G protein-coupled receptors. GRK6 is the most recently identified member of the GRK family and displays higher homology with GRK5 (70.1% amino acid identity) and IT11 (68.5%) compared to beta ARK (37.4%) and rhodopsin kinase (47.1%). To further characterize GRK6, it has been overexpressed in Sf9 cells and purified to homogeneity by sequential chromatography on SP-Sepharose and heparin-Sepharose columns. GRK6 shares a number of in vitro characteristics with GRK5, including potent inhibition by heparin and dextran sulfate (IC50 values of approximately 15 and approximately 7 nM, respectively), hyperstimulation by polycations, and preference for phosphorylation of non-acidic peptides. Rhodopsin and the beta 2-adrenergic and m2 muscarinic cholinergic receptors serve as stimulus-dependent substrates for GRK6, but with stoichiometries significantly lower than achieved by GRK5 and beta ARK. Additionally, GRK6 does not undergo significant autophosphorylation even though it contains residues identical to those that are autophosphorylated in GRK5 and rhodopsin kinase. These data extend our knowledge of a growing family of receptor-specific kinases and suggest that GRK6 has a substrate specificity distinct from beta ARK, rhodopsin kinase, and GRK5.
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PMID:Expression, purification, and characterization of the G protein-coupled receptor kinase GRK6. 807 21

G protein-coupled receptor kinases (GRKs) such as rhodopsin kinase and the beta-adrenergic receptor kinase (beta ARK) play an important role in agonist-specific phosphorylation and desensitization of G protein-coupled receptors. GRK5 is a recently identified member of the GRK family that has greater homology with rhodopsin kinase than with beta ARK. To further characterize the activity of GRK5, it has been overexpressed in Sf9 insect cells and purified by successive chromatography on S-Sepharose and Mono S columns. GRK5 phosphorylates the beta 2-adrenergic receptor (beta 2AR), m2 muscarinic cholinergic receptor, and rhodopsin in an agonist-dependent manner to maximal stoichiometries of approximately 2.5, 1.5, and 1 mol of phosphate/mol of receptor, respectively, with Km values of approximately 0.5 microM for the beta 2AR, approximately 16 microM for rhodopsin, and approximately 24 microM for ATP. Peptide phosphorylation studies suggest that in contrast to beta ARK and rhodopsin kinase, GRK5 preferentially phosphorylates on nonacidic peptides with a Km of approximately 1.5 mM. Heparin and dextran sulfate were found to be potent inhibitors of GRK5 with IC50 values of approximately 1 nM, thereby being at least 150-fold more potent on GRK5 than on beta ARK. GRK5 can also be activated by polycations, with 10 microM polylysine promoting an approximately 2.6-fold activation. Overall, these studies demonstrate that GRK5 has unique properties that distinguish it from other members of the GRK family and that likely play an important role in modulating its mechanism of action.
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PMID:Expression, purification, and characterization of the G protein-coupled receptor kinase GRK5. 828 67

G protein-coupled receptor kinases (GRK), such as the beta-adrenergic receptor kinase (beta ARK) and rhodopsin kinase, specifically phosphorylate the activated form of G protein-coupled receptors. To identify additional members of the GRK family, we screened a human heart cDNA library by low stringency hybridization using the catalytic domains of two beta ARK isoforms. Here we report the isolation of a cDNA that encodes a 576-amino-acid protein kinase, termed GRK6, that has significant homology with GRK5 (70.1% amino acid identity), IT11 (68.5%), rhodopsin kinase (47.1%), and beta ARK (37.4%). RNA blot analysis of GRK6 with selected human tissues reveals two distinct mRNAs of 3 and 2.4 kilobases with a distribution very similar to that of beta ARK (i.e. brain, skeletal muscle > pancreas > heart, lung, kidney, placenta > liver). GRK6, overexpressed in Sf9 insect cells using the baculovirus system, was able to phosphorylate both the beta 2-adrenergic receptor and rhodopsin in a stimulus-dependent fashion, although it was significantly less active then beta ARK on these substrates. These data extend the family of GRKs and suggest that GRK6 may have a substrate specificity quite distinct from beta ARK and rhodopsin kinase.
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PMID:Molecular cloning and expression of GRK6. A new member of the G protein-coupled receptor kinase family. 836 96

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.
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PMID:Feedback inhibition of G protein-coupled receptor kinase 2 (GRK2) activity by extracellular signal-regulated kinases. 1057 13

G-protein-coupled receptor kinases (GRKs) are involved in the regulation of many G-protein-coupled receptors. As opposed to the other GRKs, such as rhodopsin kinase (GRK1) or beta-adrenergic receptor kinase (beta ARK, GRK2), no receptor substrate for GRK4 has been so far identified. Here we show that GRK4 is expressed in cerebellar Purkinje cells, where it regulates mGlu(1) metabotropic glutamate receptors, as indicated by the following: 1) When coexpressed in heterologous cells (HEK293), mGlu(1) receptor signaling was desensitized by GRK4 in an agonist-dependent manner (homologous desensitization). 2) In transfected HEK293 and in cultured Purkinje cells, the exposure to glutamate agonists induced internalization of the receptor and redistribution of GRK4. There was a substantial colocalization of the receptor and kinase both under basal condition and after internalization. 3) Kinase activity was necessary for desensitizing mGlu(1a) receptor and agonist-dependent phosphorylation of this receptor was also documented. 4) Antisense treatment of cultured Purkinje cells, which significantly reduced the levels of GRK4 expression, induced a marked modification of the mGlu(1)-mediated functional response, consistent with an impaired receptor desensitization. The critical role for GRK4 in regulating mGlu(1) receptors implicates a major involvement of this kinase in the physiology of Purkinje cell and in motor learning.
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PMID:The G-protein-coupled receptor kinase GRK4 mediates homologous desensitization of metabotropic glutamate receptor 1. 1109 76


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