Dynamic Analysis of GH Receptor Conformational Changes by Split Luciferase Complementation

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 kin...

Full description

Saved in:
Bibliographic Details
Published in:Molecular endocrinology (Baltimore, Md.) Md.), 2014-11, Vol.28 (11), p.1807-1819
Main Authors: 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
Format: Article
Language:English
Subjects:
Carrier Proteins - genetics
Carrier Proteins - metabolism
Cell Line
Growth Hormone - genetics
Growth Hormone - metabolism
Humans
Janus Kinase 2 - genetics
Janus Kinase 2 - metabolism
Luciferases - metabolism
Original Research
Phosphorylation - genetics
Protein Binding - genetics
Protein Structure, Tertiary - genetics
Signal Transduction - genetics
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:0888-8809
1944-9917
DOI:10.1210/me.2014-1153