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1.
J Biol Chem ; 273(35): 22442-52, 1998 Aug 28.
Article in English | MEDLINE | ID: mdl-9712868

ABSTRACT

Prostaglandin receptors may be activated by their cognate ligand or by free radical catalyzed isoprostanes, products of arachidonic acid peroxidation. For example, prostaglandin F2alpha (PGF2alpha) causes hypertrophy of neonatal rat ventricular myocytes, via the PGF2alpha receptor (FP). However, the FP may also be activated by the isoprostane, 8,12-iso-iPF2alpha-III (Kunapuli, P., Lawson, J. A., Rokach, J., and FitzGerald, G. A. (1997) J. Biol. Chem. 272, 27147-27154). Both ligands induce myocyte hypertrophy with overlapping potencies. Interestingly, the hypertrophic effects of these two agonists on cardiomyocytes are additive. Furthermore, the preference of these two agonists for activation of intracellular signal transduction pathways differs in several respects. Thus, PGF2alpha and 8,12-iso-iPF2alpha-III stimulate inositol phosphate formation with EC50 values of 50 +/- 12 nM and 3.5 +/- 0.6 microM, respectively. Moreover, PGF2alpha causes a robust activation ( approximately 50-fold) of Erk2, whereas 8,12-iso-iPF2alpha-III has no effect. Similarly, PGF2alpha causes translocation of cytosolic phospholipase A2 and also results in a 7-fold increment in the formation of 6-keto-PGF1alpha, whereas 8,12-iso-iPF2alpha-III exerts no effect on this pathway. On the other hand, both agonists are equally potent in activating JNK1 and c-Jun, whereas neither activates the p38 kinase. Both PGF2alpha and 8,12-iso-iPF2alpha-III activate the p70S6 kinase (p70(S6K)), but not Akt, downstream of phosphatidylinositol-3-kinase (PI3K). However, both wortmannin, a PI3K inhibitor, and rapamycin, an inhibitor of p70(S6K) activity, inhibit 8,12-iso-iPF2alpha-III -induced myocyte hypertrophy, with IC50 values of 60 +/- 12 and 3 +/- 1.7 nM, respectively, whereas neither compound abrogates the PGF2alpha-mediated response. Thus, both PGF2alpha and 8,12-iso-iPF2alpha-III induce myocyte hypertrophy via discrete signaling pathways. Although both agonists signal via the JNK pathway to initiate changes in c-Jun-dependent gene transcription, PGF2alpha preferentially activates the MEK-Erk2- cytosolic phospholipase A2 pathway. In contrast, the PI3K-p70(S6K) pathway appears to be essential for 8,12-iso-iPF2alpha-III-induced myocyte hypertrophy.


Subject(s)
Cardiomegaly/chemically induced , Dinoprost/analogs & derivatives , Dinoprost/pharmacology , Signal Transduction/drug effects , Animals , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cells, Cultured , Enzyme Activation , Phospholipases A/metabolism , Phospholipases A2 , Rats , Rats, Sprague-Dawley , Substrate Specificity
2.
J Biol Chem ; 272(43): 27147-54, 1997 Oct 24.
Article in English | MEDLINE | ID: mdl-9341156

ABSTRACT

Prostaglandin F2alpha (PGF2alpha) is a product of cyclooxygenase-catalyzed metabolism of arachidonic acid. Recently, PGF2alpha analogs have been hypothesized to reduce intraocular pressure via relaxation of the ciliary muscle. To investigate the molecular basis of PGF2alpha receptor (FP) activation in the eye, we cloned the FP from a human ciliary body (hcb) cDNA library. The open reading frame of the hcb-FP cDNA was identical to the uterine FP cDNA. The hcb-FP appeared to be predominantly membrane-localized, as visualized by an FP-specific peptide antibody, and coupled to inositol phosphate formation when stably expressed in HEK 293 cells. Interestingly, the hcb-FP could also be activated by the F2 isoprostane, 12-iso-PGF2alpha, in addition to its cognate ligand, PGF2alpha. 12-iso-PGF2alpha was less potent (EC50 = 5 microM) than PGF2alpha (EC50 = 10 nM) in generating inositol phosphates via the hcb-FP in HEK 293 cells. Both ligands also stimulated mitogenesis in NIH 3T3 cells. Although 12-iso-PGF2alpha caused a dose-dependent activation of the FP, it failed to activate the recombinant human prostacyclin receptor and caused only minimal activation of the thromboxane receptor isoforms stably expressed in HEK 293 cells. Four additional F2 isoprostanes, 8-iso-PGF2alpha, IPF2alpha-I, IPF2alpha-III, and 9beta,11beta-PGF2, caused trivial, or no, activation of the FP. Consistent with these observations, only PGF2alpha and 12-iso-PGF2alpha caused rapid homologous desensitization of FP and also exhibited cross-desensitization, with PGF2alpha resulting in a maximum of approximately 60% desensitization. The human FP may thus be activated specifically, by the free radical-catalyzed F2 isoprostane, 12-iso-PGF2alpha, in addition to the cyclooxygenase product, PGF2alpha. Incidental receptor activation by isoprostanes may complement the actions of PGF2alpha in clinical syndromes where oxidant stress and augmented prostaglandin biosynthesis coincide.


Subject(s)
Ciliary Body/metabolism , Dinoprost/pharmacology , Dinoprostone/analogs & derivatives , Receptors, Prostaglandin/physiology , Transcription, Genetic , 3T3 Cells , Animals , Base Sequence , Cell Division/drug effects , Cell Line , DNA Primers , DNA, Complementary , Dinoprostone/pharmacology , Female , Gene Library , Humans , Inositol Phosphates/metabolism , Kinetics , Male , Mice , Molecular Sequence Data , Organ Specificity , Polymerase Chain Reaction , Pregnancy , RNA, Messenger/biosynthesis , Receptors, Prostaglandin/biosynthesis , Recombinant Proteins/metabolism , Transfection
3.
J Biol Chem ; 271(9): 5049-58, 1996 Mar 01.
Article in English | MEDLINE | ID: mdl-8617782

ABSTRACT

The alpha1B-adrenergic receptor (alpha1BAR), its truncated mutant T368, different G protein-coupled receptor kinases (GRK) and arrestin proteins were transiently expressed in COS-7 or HEK293 cells alone and/or in various combinations. Coexpression of beta-adrenergic receptor kinase (betaARK) 1 (GRK2) or 2 (GRK3) could increase epinephrine-induced phosphorylation of the wild type alpha1BAR above basal as compared to that of the receptor expressed alone. On the other hand, overexpression of the dominant negative betaARK (K220R) mutant impaired agonist-induced phosphorylation of the receptor. Overexpression of GRK6 could also increase epinephrine-induced phosphorylation of the receptor, whereas GRK5 enhanced basal but not agonist-induced phosphorylation of the alpha1BAR. Increasing coexpression of betaARK1 or betaARK2 resulted in the progressive attenuation of the alpha1BAR-mediated response on polyphosphoinositide (PI) hydrolysis. However, coexpression of betaARK1 or 2 at low levels did not significantly impair the PI response mediated by the truncated alpha1BAR mutant T368, lacking the C terminus, which is involved in agonist-induced desensitization and phosphorylation of the receptor. Similar attenuation of the receptor-mediated PI response was also observed for the wild type alpha1BAR, but not for its truncated mutant, when the receptor was coexpressed with beta-arrestin 1 or beta-arrestin 2. Despite their pronounced effect on phosphorylation of the alpha1BAR, overexpression of GRK5 or GRK6 did not affect the receptor-mediated response. In conclusion, our results provide the first evidence that betaARK1 and 2 as well as arrestin proteins might be involved in agonist-induced regulation of the alpha1BAR. They also identify the alpha1BAR as a potential phosphorylation substrate of GRK5 and GRK6. However, the physiological implications of GRK5- and GRK6-mediated phosphorylation of the alpha1BAR remain to be elucidated.


Subject(s)
Adrenergic beta-Agonists/pharmacology , Arrestins , Cyclic AMP-Dependent Protein Kinases/metabolism , Epinephrine/pharmacology , GTP-Binding Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Adrenergic, alpha-1/physiology , Animals , Antigens/biosynthesis , Antigens/isolation & purification , Antigens/metabolism , Blotting, Western , Cattle , Cell Line , Chlorocebus aethiops , Cyclic AMP-Dependent Protein Kinases/biosynthesis , Eye Proteins/biosynthesis , Eye Proteins/isolation & purification , Eye Proteins/metabolism , GTP-Binding Proteins/biosynthesis , GTP-Binding Proteins/isolation & purification , Gene Expression , Humans , Kidney , Kinetics , Mutagenesis , Phosphatidylinositols/metabolism , Phosphorylation , Receptor Protein-Tyrosine Kinases/biosynthesis , Receptor Protein-Tyrosine Kinases/isolation & purification , Receptors, Adrenergic, alpha-1/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Transfection , beta-Adrenergic Receptor Kinases , beta-Arrestin 1 , beta-Arrestin 2 , beta-Arrestins
4.
Mol Pharmacol ; 47(2): 224-33, 1995 Feb.
Article in English | MEDLINE | ID: mdl-7870029

ABSTRACT

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.


Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , GTP-Binding Proteins/metabolism , Muscarinic Agonists/pharmacology , Receptors, Muscarinic/metabolism , Animals , Baculoviridae/genetics , Cell Line , Cell Membrane/metabolism , Humans , Kinetics , Phosphorylation , Receptors, Muscarinic/genetics , Spodoptera , Substrate Specificity , beta-Adrenergic Receptor Kinases
5.
Cytogenet Cell Genet ; 70(3-4): 250-4, 1995.
Article in English | MEDLINE | ID: mdl-7789183

ABSTRACT

G protein-coupled receptor kinases (GRKs) play an important role in phosphorylating and regulating the activity of a variety of G protein-coupled receptors. Chromosomal mapping of the human genes for the two most recently identified members of the GRK family, GRK5 (GPRK5) and GRK6 (GPRK6), was accomplished by correlation of the presence of the GPRK5 and GPRK6 loci with specific chromosome regions in a rodent-human hybrid panel. These analyses revealed that GPRK5 maps to chromosome region 10q24-->qter while GPRK6 maps to 5q35. A GPRK6 related locus maps to 13pter-->q21.


Subject(s)
Chromosomes, Human, Pair 10 , Chromosomes, Human, Pair 5 , Protein Serine-Threonine Kinases , Receptor Protein-Tyrosine Kinases/genetics , Animals , Base Sequence , Chromosome Mapping , Cricetinae , Cricetulus , DNA Primers , Female , G-Protein-Coupled Receptor Kinase 5 , G-Protein-Coupled Receptor Kinases , GTP-Binding Proteins/genetics , Humans , Hybrid Cells , Molecular Sequence Data , Open Reading Frames , Placenta/metabolism , Polymerase Chain Reaction , Pregnancy
6.
J Biol Chem ; 269(14): 10209-12, 1994 Apr 08.
Article in English | MEDLINE | ID: mdl-8144599

ABSTRACT

G protein-coupled receptor kinases (GRKs) play an important role in mediating agonist-specific desensitization of numerous G protein-coupled receptors. GRK5, a recently identified member of the GRK family, undergoes a rapid phospholipid-stimulated autophosphorylation to a stoichiometry of approximately 2 mol of phosphate/mol of GRK5. The ability of phospholipids to stimulate autophosphorylation is largely blocked by a glutathione S-transferase fusion protein containing the last 102 amino acids of GRK5 (amino acids 489-590), suggesting that this is a primary region involved in GRK5/phospholipid interaction. Phosphoamino acid determination and mutagenesis studies demonstrate that autophosphorylation of GRK5 occurs primarily at residues Ser-484 and Thr-485. Expression and characterization of a mutant GRK5 that does not autophosphorylate (S484A and T485A) reveals that the mutant has a approximately 15-20-fold reduced ability to phosphorylate the beta 2-adrenergic receptor and rhodopsin compared to wild type GRK5. These results suggest that phospholipid-stimulated autophosphorylation may represent a novel mechanism for membrane association and regulation of GRK5 activity.


Subject(s)
GTP-Binding Proteins/metabolism , Phospholipids/metabolism , Protein Serine-Threonine Kinases , Receptor Protein-Tyrosine Kinases/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cells, Cultured , Cricetinae , DNA Primers , G-Protein-Coupled Receptor Kinase 5 , Lepidoptera , Molecular Sequence Data , Phosphorylation
7.
J Biol Chem ; 269(2): 1099-105, 1994 Jan 14.
Article in English | MEDLINE | ID: mdl-8288567

ABSTRACT

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.


Subject(s)
GTP-Binding Proteins/metabolism , Protein Serine-Threonine Kinases , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Cell Surface/metabolism , Amino Acid Sequence , Cations, Divalent , G-Protein-Coupled Receptor Kinase 5 , Humans , Kinetics , Molecular Sequence Data , Peptides/chemistry , Peptides/metabolism , Protein Kinases/metabolism , Recombinant Proteins , Substrate Specificity
8.
Proc Natl Acad Sci U S A ; 90(12): 5588-92, 1993 Jun 15.
Article in English | MEDLINE | ID: mdl-7685906

ABSTRACT

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.


Subject(s)
Eye Proteins , GTP-Binding Proteins/biosynthesis , Gene Expression , Multigene Family , Protein Kinases/biosynthesis , Protein Serine-Threonine Kinases , Protein-Tyrosine Kinases/biosynthesis , RNA, Messenger/metabolism , Receptors, Cell Surface/biosynthesis , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Cattle , Cell Line , Cloning, Molecular , G-Protein-Coupled Receptor Kinase 1 , G-Protein-Coupled Receptor Kinase 5 , Humans , Macromolecular Substances , Molecular Sequence Data , Moths , Oligodeoxyribonucleotides , Organ Specificity , Poly A/analysis , Poly A/metabolism , Polymerase Chain Reaction , Protein Kinases/genetics , RNA/analysis , RNA/metabolism , RNA, Messenger/analysis , Rod Cell Outer Segment/metabolism , Sequence Homology, Amino Acid , Transfection
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