ABSTRACT
(2-Hydroxyethyl) dimethylsulfoxonium chloride (1), the well-known causative agent of Dogger Bank Itch, has been isolated as a cytotoxic constituent from the marine sponge Theonella aff. mirabilis. The structure of 1 was determined by spectral and X-ray crystallographic analyses.
Subject(s)
Chlorides/isolation & purification , Dermatitis, Contact/etiology , Porifera/chemistry , Sulfonium Compounds/isolation & purification , Animals , Chlorides/chemistry , Chlorides/pharmacology , Crystallography, X-Ray , Cytotoxins/chemistry , Inhibitory Concentration 50 , Magnetic Resonance Spectroscopy , Mice , Models, Chemical , Models, Molecular , Sulfonium Compounds/chemistry , Sulfonium Compounds/pharmacology , Tumor Cells, CulturedABSTRACT
Substance P receptor (SPR), which plays a key role in pain transmission, is known to undergo rapid agonist-dependent desensitization and internalization. The present study shows that human SPR undergoes agonist-dependent phosphorylation in intact cells. Immunoprecipitation of SPR from 32Pi-labeled Chinese hamster ovary cells stably expressing human SPR (CHO-hSPR) indicates that substance P (SP) causes a rapid (T1/2 < 1 min), dose-dependent (EC50 = 2 nM), and pronounced (5-fold over basal) phosphorylation of SPR. Because SPR in CHO-hSPR couples to Galphaq, Galphas, and Galphao (), we examined the involvement of various second messenger-activated protein kinases in SPR phosphorylation. Although increases in intracellular cyclic AMP or treatment with the calcium ionophore A23187 do not cause SPR phosphorylation, treatment with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) causes a 2.5-fold increase in SPR phosphorylation with a T1/2 of <1 min. However, PKC inhibitor GF109203X has no effect on SP-dependent SPR phosphorylation. Furthermore, although SP treatment phosphorylates SPR on both serine and threonine residues equally, PMA treatment phosphorylates the receptor predominantly on serine residues. Two-dimensional phosphopeptide mapping data indicate that SP-dependent and PMA-dependent phosphorylations of SPR have some unique differences. Taken together, these data suggest that although activation of PKC by PMA can lead to SPR phosphorylation, PKC does not mediate SP-dependent phosphorylation of SPR. In conclusion, the present study represents the first demonstration and characterization of agonist-dependent and PMA-mediated phosphorylation of SPR in intact cells.
Subject(s)
Receptors, Neurokinin-1/metabolism , Animals , CHO Cells , Cricetinae , Humans , Phosphorylation , Protein Kinase C/physiology , Receptors, Neurokinin-1/agonists , Receptors, Neurokinin-1/drug effects , Receptors, Neurokinin-1/genetics , Substance P/metabolism , Tetradecanoylphorbol Acetate/pharmacology , TransfectionABSTRACT
The substance P receptor (SPR) is a G protein-coupled receptor (GPCR) that plays a key role in pain regulation. The SPR desensitizes in the continued presence of agonist, presumably via mechanisms that implicate G protein-coupled receptor kinases (GRKs) and beta-arrestins. The temporal relationship of these proposed biochemical events has never been established for any GPCR other than rhodopsin beyond the resolution provided by biochemical assays. We investigate the real-time activation and desensitization of the human SPR in live HEK293 cells using green fluorescent protein conjugates of protein kinase C, GRK2, and beta-arrestin 2. The translocation of protein kinase C betaII-green fluorescent protein to and from the plasma membrane in response to substance P indicates that the human SPR becomes activated within seconds of agonist exposure, and the response desensitizes within 30 s. This desensitization process coincides with a redistribution of GRK2 from the cytosol to the plasma membrane, followed by a robust redistribution of beta-arrestin 2 and a profound change in cell morphology that occurs after 1 min of SPR stimulation. These data establish a role for GRKs and beta-arrestins in homologous desensitization of the SPR and provide the first visual and temporal resolution of the sequence of events underlying homologous desensitization of a GPCR in living cells.
Subject(s)
Arrestins/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Receptors, Neurokinin-1/metabolism , Biological Transport , Cell Line , Cell Membrane/enzymology , Cytoplasm/enzymology , Humans , Neurokinin-1 Receptor Antagonists , Protein Kinase C/metabolism , Receptors, Neurokinin-1/agonists , Substance P/pharmacology , beta-Adrenergic Receptor Kinases , beta-Arrestin 2 , beta-ArrestinsABSTRACT
G protein-coupled receptor kinases (GRKs) phosphorylate agonist-occupied G protein-coupled receptors (GPCRs), resulting in GPCR desensitization. GRK2 is one of the better studied of the six known GRKs and phosphorylates several GPCRs. In a previous study, we documented that GRK2 and GRK3 phosphorylate purified and reconstituted rat substance P receptor (rSPR) [Kwatra et al. (1993) J. Biol. Chem. 268, 9161-9164]. Here, we characterize in detail GRK2-catalyzed phosphorylation of human SPR (hSPR) in intact membranes. GRK2 phosphorylates hSPR in urea-washed Sf9 membranes in an agonist-dependent manner with a stoichiometry of 19 +/- 1 mol of phosphate/mol of receptor, which increases slightly (1.3-fold increase) in the presence of G beta gamma. Kinetic analyses indicate that receptor phosphorylation occurs with a Km of 6.3 +/- 0.4 nM and a Vmax of 1.8 +/- 0.1 nmol/min/mg; these kinetic parameters are only slightly affected by G beta gamma [Km = 3.6 +/- 1.0 nM and Vmax = 2.2 +/- 0.2 nmol/min/mg]. The lack of a strong stimulatory effect of G beta gamma on GRK2-catalyzed phosphorylation of hSPR is surprising since G beta gamma potently stimulates GRK2-catalyzed phosphorylation of beta 2-adrenergic receptor and rhodopsin. Involvement of G beta gamma endogenously present in membranes is ruled out as a source of high levels of hSPR phosphorylation, since receptor phosphorylation was not affected by guanine nucleotides that suppress or enhance the release of endogenous G beta gamma. The present study determines, for the first time, the kinetics of phosphorylation of a receptor substrate of GRK2 in intact membranes. Further, our results identify hSPR as a unique substrate of GRK2 whose phosphorylation is strong even in the absence of G beta gamma.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Receptors, Neurokinin-1/metabolism , Amino Acid Sequence , Animals , Catalysis/drug effects , Cell Membrane , GTP-Binding Proteins/metabolism , GTP-Binding Proteins/physiology , Humans , Kinetics , Molecular Sequence Data , Phosphorylation/drug effects , Protein Binding/drug effects , Receptors, Neurokinin-1/agonists , Receptors, Neurokinin-1/drug effects , Spodoptera , Urea , beta-Adrenergic Receptor KinasesABSTRACT
A 60-year-old man with severe DCM was scheduled for a herniorrhaphy under epidural anesthesia using fentanyl. Three months prior to operation, the patient suffered heart failure associated with life-threatening ventricular arrhythmia. The former was successfully treated with pimobendan as the main constituent of medication, but the latter was not responsive to various antiarrhythmic drugs with the exception of aprindine. On the day of operation and for two days postoperatively, pimobendan was administered daily. A sudden drop in systemic blood pressure and central venous pressure (CVP) during anesthesia, as well as the tendency to hypotensive status in the postoperative period were well regulated with continuous infusion of dopamine and dobutamine via CVP catheter probably due to the effect of up-regulation of pimobendan, together with adjustment of the volume of intravenous fluids. No dangerous ventricular arrhythmia were observed. Thereafter the patient made uneventful progress and was discharged on the 8th postoperative day.
