RESUMO
The eukaryotic RNA polymerases Pol I, Pol II, and Pol III are the central multiprotein machines that synthesize ribosomal, messenger, and transfer RNA, respectively. Here we provide a catalog of available structural information for these three enzymes. Most structural data have been accumulated for Pol II and its functional complexes. These studies have provided insights into many aspects of the transcription mechanism, including initiation at promoter DNA, elongation of the mRNA chain, tunability of the polymerase active site, which supports RNA synthesis and cleavage, and the response of Pol II to DNA lesions. Detailed structural studies of Pol I and Pol III were reported recently and showed that the active center region and core enzymes are similar to Pol II and that strong structural differences on the surfaces account for gene class-specific functions.
Assuntos
RNA Polimerases Dirigidas por DNA , Modelos Químicos , Modelos Genéticos , RNA/biossíntese , RNA/química , Transcrição Gênica/genética , Animais , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/ultraestrutura , Ativação Enzimática , Humanos , Modelos Moleculares , RNA/genéticaRESUMO
beta-arrestin is a cytosolic protein thought to be responsible for uncoupling agonist-activated beta 2-adrenergic receptors from their guanine-nucleotide-binding proteins (G-protein) subsequent to receptor phosphorylation by the beta-adrenergic receptor kinase (beta ARK). In order to investigate this interaction, we generated a recombinant baculovirus for the expression of beta-arrestin in Sf9 insect cells. Apparently homogeneous beta-arrestin preparations were obtained in a one-step purification on heparin-Sepharose. Purified beta-arrestin bound to rhodopsin in a phosphorylation-dependent plus light-dependent manner. Binding to beta 2-adrenergic receptors was investigated using purified receptors reconstituted into lipid vesicles. The accessibility of the reconstituted receptors was determined using the agonist isoproterenol for the ligand-binding site and an antibody binding to an attached myc tag for the C-terminus, the site of receptor phosphorylation. On the basis of these data, the binding of purified beta-arrestin to beta ARK-phosphorylated beta 2-adrenergic receptors was found to occur with a KD of 1.8 nM and with a maximum of 1 beta-arrestin/receptor. beta-arrestin also bound to receptors which had been completely dephosphorylated with acid phosphatase, but the affinity was approximately 30-fold lower. In contrast to regulation by phosphorylation, binding of agonists or antagonists to the receptors had negligible effects on beta-arrestin binding. Finally, beta-arrestin and beta ARK were shown to be capable of producing synergistic inhibition of beta 2-adrenergic-receptor-stimulated adenylyl cyclase activity of cell membranes. These data show that high-affinity stoichiometric binding of beta-arrestin to beta 2-adrenergic receptors occurs in a beta ARK-dependent manner and is sufficient to impair adenylyl cyclase stimulation by the receptors.
Assuntos
Antígenos/metabolismo , Arrestinas , Proteínas do Olho/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Adenilil Ciclases/metabolismo , Animais , Antígenos/genética , Bovinos , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas do Olho/genética , Expressão Gênica , Humanos , Cinética , Fosforilação , Ligação Proteica , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rodopsina/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Spodoptera , Quinases de Receptores Adrenérgicos beta , beta-ArrestinasRESUMO
The beta-adrenergic receptor kinase mediates agonist-dependent phosphorylation of beta-adrenergic receptors, which is thought to represent the first step of homologous desensitization. We have expressed bovine and human beta ARK1 in Sf9 cells and purified them to apparent homogeneity in milligram quantities. The Km-values of the enzyme were 3.8 microM for rhodopsin and 22 microM for ATP; the Vmax-value was 9.9 mol phosphate/mol beta ARK/min. These data indicate that the two recombinant kinases were at least as active as preparations previously obtained from bovine brain. There were no differences in the functional activity of human and bovine beta ARK.
