ABSTRACT
beta-Arrestins are proteins that bind phosphorylated heterotrimeric GTP-binding protein (G protein)-coupled receptors (GPCRs) and contribute to the desensitization of GPCRs by uncoupling the signal transduction process. Resensitization of GPCR responsiveness involves agonist-mediated receptor sequestration. Overexpression of beta-arrestins in human embryonic kidney cells rescued the sequestration of beta 2-adrenergic receptor (beta 2AR) mutants defective in their ability to sequester, an effect enhanced by simultaneous overexpression of beta-adrenergic receptor kinase 1. Wild-type beta 2AR sequestration was inhibited by the overexpression of two beta-arrestin mutants. These findings suggest that beta-arrestins play an integral role in GPCR internalization and thus serve a dual role in the regulation of GPCR function.
Subject(s)
Adrenergic beta-Agonists/pharmacology , Antigens/physiology , Arrestins , Cyclic AMP-Dependent Protein Kinases/metabolism , Eye Proteins/physiology , GTP-Binding Proteins/metabolism , Receptors, Adrenergic, beta-2/metabolism , Antigens/genetics , Cell Line , Cyclic AMP-Dependent Protein Kinases/genetics , DNA, Complementary , Eye Proteins/genetics , Humans , Isoproterenol/pharmacology , Mutation , Phosphorylation , Point Mutation , Receptors, Adrenergic, beta-2/genetics , Transfection , beta-Adrenergic Receptor Kinases , beta-ArrestinsABSTRACT
Post-transcriptional regulation of genes important in iron metabolism, ferritin and the transferrin receptor (TfR), is achieved through regulated binding of a cytosolic protein, the iron-responsive element binding protein (IRE-BP), to RNA stem-loop motifs known as iron-responsive elements (IREs). Binding of the IRE-BP represses ferritin translation and represses degradation of the TfR mRNA. The IRE-BP senses iron levels and accordingly modifies binding to IREs through a novel sensing mechanism. An iron-sulfur cluster of the IRE-BP reversibly binds iron; when cytosolic iron levels are depleted, the cluster becomes depleted of iron and the IRE-BP acquires the capacity to bind IREs. When cytosolic iron levels are replete, the IRE-BP loses RNA binding capacity, but acquires enzymatic activity as a functional aconitase. RNA binding and aconitase activity are mutually exclusive activities of the IRE-BP, and the state of the iron-sulfur cluster determines how the IRE-BP will function.
Subject(s)
Ferritins/genetics , Gene Expression Regulation , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Receptors, Transferrin/genetics , Aconitate Hydratase/metabolism , Amino Acid Sequence , Animals , Humans , Iron/metabolism , Iron-Regulatory Proteins , Molecular Sequence Data , RNA-Binding Proteins/chemistry , Receptors, Transferrin/metabolism , Sulfur/metabolismABSTRACT
A clone for the iron-responsive element (IRE)-binding protein (IRE-BP) has been transfected and expressed in mouse fibroblasts. The IRE-BP gene product binds IREs with high affinity and specificity. Amino acid alignments reveal that the IRE-BP is 30% identical to mitochondrial aconitase. The 18 active site residues of mitochondrial aconitase are identical to those in the IRE-BP, suggesting that the IRE-BP may possess aconitase activity. After purification of native IRE-BP and immunoaffinity purification of transfected and expressed IRE-BP, we demonstrate that the purified IRE-BP has aconitase activity.
Subject(s)
Aconitate Hydratase/metabolism , RNA-Binding Proteins/metabolism , Aconitate Hydratase/genetics , Amino Acid Sequence , Animals , Cells, Cultured , Chimera , Cloning, Molecular , DNA/genetics , Ferritins/metabolism , Genes, myc , Humans , Iron-Regulatory Proteins , Mice , Molecular Sequence Data , Open Reading Frames , RNA-Binding Proteins/genetics , Restriction Mapping , Transcription, Genetic , TransfectionABSTRACT
Pretransplant donor-specific blood transfusion (DSBT) has been shown to enhance renal allograft survival in man and indefinitely prolong renal transplants among various MHC-disparate rat strains. Using PVG (RT1c) recipients and ACI (RT1a) donor-strain rats, DSBT alone was found to elicit complement-dependent cytotoxic IgM antibody (Ab) to donor class I (RT1.Aa) alloantigens that peaked at 7 days. An enzyme-linked immunosorbent assay was developed to measure host Ab against allospecific (idiotypic) determinants on the anti-RT1.Aa monoclonal Ab R2/10P, R2/15S, and YR1/100. Following DSBT alone, antiidiotypic Ab were detected in the circulation within 7-11 days after transfusion. Transplantation of a donor strain kidney in the presence of antiidiotypic Ab at day 7 or 11 post-DSBT resulted in enhanced graft survival and a rapid decline in circulating alloantibody, such that by days 4-6 posttransplantation little IgM or IgG alloantibody was detected. In contrast, all 6 PVG rats that were transplanted 4 days after DSBT (prior to development of detectable antiidiotypic Ab) rejected their grafts within 30 days, and 4 of 6 showed elevated alloantibody titers within 3 days posttransplantation. Control PVG rats receiving autologous blood transfusion (ABT) alone developed no alloantibody response but developed high titers of donor-specific alloantibody by 6 days posttransplantation, at the time of irreversible rejection. ABT alone did not elicit antiidiotypic Ab and ABT pretreated graft recipients developed antiidiotypic Ab only after the onset of rejection at day 4. In both DSBT and ABT groups, the antiidiotypic Ab were primarily IgM, IgG1, and IgG2c. These findings indicate that DSBT induces production of cytotoxic alloantibodies followed by an antiidiotypic Ab response at days 7-11, during which time transplanted renal allografts are not rejected and there is a reduction in circulating alloantibody. In contrast, renal allografts placed in DSBT-treated rats prior to antiidiotypic Ab development (less than or equal to 4 days) or in ABT-treated rats that do not develop any antiidiotypic Ab, elicit a rapid rise in alloantibody and are rejected.
