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Dynamic analysis of GH receptor conformational changes by split luciferase complementation.
Liu, Ying; Berry, Philip A; Zhang, Yue; Jiang, Jing; Lobie, Peter E; Paulmurugan, Ramasamy; Langenheim, John F; Chen, Wen Y; Zinn, Kurt R; Frank, Stuart J.
Afiliação
  • Liu Y; Department of Medicine (Y.L., P.A.B., Y.Z., J.J., S.J.F.), Division of Endocrinology, Diabetes, and Metabolism, and Departments of Radiology (K.R.Z.), and Cell, Developmental, and Integrative Biology (S.J.F.), University of Alabama at Birmingham, Birmingham, Alabama 35294; Cancer Science Institute of Singapore and Department of Pharmacology (P.E.L.), National University of Singapore, Singapore 119077; Department of Radiology (R.P.), Stanford University School of Medicine, Palo Alto, California 9
Mol Endocrinol ; 28(11): 1807-19, 2014 Nov.
Article em En | MEDLINE | ID: mdl-25188449
The transmembrane GH receptor (GHR) exists at least in part as a preformed homodimer on the cell surface. Structural and biochemical studies suggest that GH binds GHR in a 1:2 stoichiometry to effect acute GHR conformational changes that trigger the activation of the receptor-associated tyrosine kinase, Janus kinase 2 (JAK2), and downstream signaling. Despite information about GHR-GHR association derived from elegant fluorescence resonance energy transfer/bioluminescence resonance energy transfer studies, an assessment of the dynamics of GH-induced GHR conformational changes has been lacking. To this end, we used a split luciferase complementation assay that allowed detection in living cells of specific ligand-independent GHR-GHR interaction. Furthermore, GH treatment acutely augmented complementation of enzyme activity between GHRs fused, respectively, to N- and C-terminal fragments of firefly luciferase. Analysis of the temporal pattern of GH-induced complementation changes, pharmacological manipulation, genetic alteration of JAK2 levels, and truncation of the GHR intracellular domain (ICD) tail suggested that GH acutely enhances proximity of the GHR homodimer partners independent of the presence of JAK2, phosphorylation of GHR-luciferase chimeras, or an intact ICD. However, subsequent reduction of complementation requires JAK2 kinase activity and the ICD tail. This conclusion is in contrast to existing models of the GHR activation process.
Assuntos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Proteínas de Transporte / Estrutura Terciária de Proteína / Luciferases Limite: Humans Idioma: En Revista: Mol Endocrinol Assunto da revista: BIOLOGIA MOLECULAR / ENDOCRINOLOGIA Ano de publicação: 2014 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Proteínas de Transporte / Estrutura Terciária de Proteína / Luciferases Limite: Humans Idioma: En Revista: Mol Endocrinol Assunto da revista: BIOLOGIA MOLECULAR / ENDOCRINOLOGIA Ano de publicação: 2014 Tipo de documento: Article