Construction of a soluble human GH-receptor/EGF-receptor hybrid and its activation by GH.
To develop a cell-free system that can be used to measure cytokine bioactivity we have designed a soluble hybrid molecule consisting of the extracellular domain of the GH-receptor (GHR) and the intracellular domain of the epidermal growth factor receptor (EGFR). A DNA construct encoding this hybrid-receptor was inserted into a baculoviral expression vector and expressed in Sf9-cells. Activation of the hybrid-receptor by ligand-induced dimerization can be measured as the incorporation of radiolabeled phosphate into a biotinylated tyrosine kinase peptide substrate. The kinase activity in samples stimulated with GH (10 ng/ml) increased 5-fold compared to samples without addition of GH. This is the first example of a functional hybrid-receptor where the transmembrane domain has been deleted. Our results suggest that such hybrid-receptors may be used for detection of GH and other cytokine-receptor activating substances in biological fluids.
Hellgren
G
G
Göteborg Pediatric Growth Research Centre, Department of Pediatrics, The Sahlgrenska Academy at Göteborg University, Göteborg, Sweden.
Löfqvist
C
C
Wikland
K Albertsson
KA
Carlsson
B
B
Carlsson
L M S
LM
D001483:Base Sequence; D017931:DNA Primers; D066246:ErbB Receptors / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013006:Growth Hormone / Q000494:pharmacology*; D006801:Humans; D011986:Receptors, Somatotropin / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D011986:Receptors, Somatotropin; D011993:Recombinant Fusion Proteins; D013006:Growth Hormone; D066246:ErbB Receptors