Abstract
Like Ras, farnesylation of the IP (prostacyclin receptor) is required for its efficient intracellular signalling, and hence the IP represents a potential target for inhibition by FTIs [FTase (farnesyl protein transferase) inhibitors]. Herein, the effect of SCH66336 on the isoprenylation and function of the human and mouse IPs overexpressed in human embryonic kidney 293 cells, and by the IP endogenously expressed in human erythroleukaemia cells, was investigated. SCH66336 yielded concentration-dependent decreases in IP-mediated cAMP generation (IC50 0.27-0.62 nM), [Ca2+]i mobilization (IC50 26.6-48.3 nM) and IP internalization, but had no effect on signalling by the non-isoprenylated beta2 adrenergic receptor or b isoform of the TP (prostanoid thromboxane A2 receptor). Additionally, SCH66336 impaired IP-mediated crossdesensitization of TPa signalling (IC50 56.1 nM) and reduced farnesylation of the molecular chaperone protein HDJ-2 (IC50 3.1 nM). To establish whether farnesylation of the IP is inhibited and/or whether its 'CaaX motif' might undergo alternative geranylgeranylation in the presence of SCH66336, a series of chimaeric Ha (Harvey)-Ras fusions were generated by replacing its CaaX motif (-CVLS) with that of the IP (-CSLC) or, as controls, of Ki (Kirsten)-Ras 4B (-CVIM) or Rac 1 (-CVLL). Whereas SCH66336 had no effect on Ha-RasCVLL isoprenylation in vitro or in whole cells, it supported alternative geranylgeranylation of Ha-RasCVIM, but completely impaired isoprenylation of both Ha-RasCVLS and Ha-RasCSLC. These data confirm that the -CSLC motif of the IP is a direct target for inhibition by the FTI SCH66336, and in the presence of strong FTase inhibition, the IP does not undergo compensatory geranylgeranylation
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adrenergic beta-Agonists / pharmacology
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Alkyl and Aryl Transferases / antagonists & inhibitors*
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Amino Acid Motifs
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Animals
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Calcium Signaling / drug effects
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Carrier Proteins / metabolism
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Cell Line
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Cell Line, Tumor / metabolism
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Cyclic AMP / biosynthesis
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Dose-Response Relationship, Drug
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Endocytosis / drug effects
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Epoprostenol / analogs & derivatives*
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Epoprostenol / pharmacology
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Farnesyltranstransferase
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HSP40 Heat-Shock Proteins
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Heat-Shock Proteins / metabolism
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Humans
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Iloprost / metabolism
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Isoproterenol / pharmacology
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Kidney
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Leukemia, Erythroblastic, Acute / pathology
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Mice
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Mutagenesis, Site-Directed
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Organophosphorus Compounds / metabolism
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Piperidines / pharmacology*
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Proline / analogs & derivatives*
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Proline / metabolism
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Propanolamines / metabolism
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Protein Prenylation / drug effects
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Protein Processing, Post-Translational / drug effects*
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Proto-Oncogene Proteins p21(ras) / chemistry
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Proto-Oncogene Proteins p21(ras) / metabolism*
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Pyridines / pharmacology*
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Receptors, Adrenergic, beta-2 / drug effects
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Receptors, Adrenergic, beta-2 / genetics
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Receptors, Epoprostenol / drug effects*
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Receptors, Epoprostenol / metabolism
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Receptors, Thromboxane A2, Prostaglandin H2 / drug effects
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Recombinant Fusion Proteins / metabolism
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Signal Transduction / drug effects*
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Transfection
Substances
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Adrenergic beta-Agonists
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Carrier Proteins
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DNAJA1 protein, human
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HSP40 Heat-Shock Proteins
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Heat-Shock Proteins
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Organophosphorus Compounds
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Piperidines
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Propanolamines
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Pyridines
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Receptors, Adrenergic, beta-2
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Receptors, Epoprostenol
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Receptors, Thromboxane A2, Prostaglandin H2
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Recombinant Fusion Proteins
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Proline
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Epoprostenol
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Cyclic AMP
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Alkyl and Aryl Transferases
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Farnesyltranstransferase
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HRAS protein, human
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Proto-Oncogene Proteins p21(ras)
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lonafarnib
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Iloprost
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Isoproterenol
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cicaprost
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CGP 12177
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ceronapril