Dynamic analysis of GH receptor conformational changes by split luciferase complementation. Mol Endocrinol 2014 Nov;28(11):1807-19
Date
09/05/2014Pubmed ID
25188449Pubmed Central ID
PMC4213363DOI
10.1210/me.2014-1153Scopus ID
2-s2.0-84908349618 (requires institutional sign-in at Scopus site) 10 CitationsAbstract
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.
Author List
Liu Y, Berry PA, Zhang Y, Jiang J, Lobie PE, Paulmurugan R, Langenheim JF, Chen WY, Zinn KR, Frank SJMESH terms used to index this publication - Major topics in bold
Carrier ProteinsCell Line
Growth Hormone
Humans
Janus Kinase 2
Luciferases
Phosphorylation
Protein Binding
Protein Structure, Tertiary
Signal Transduction
