Construction of a soluble human GH-receptor/EGF-receptor hybrid and its activation by GH

Cytokine. 2004 Mar 21;25(6):260-4. doi: 10.1016/j.cyto.2003.11.010.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • DNA Primers
  • ErbB Receptors / drug effects
  • ErbB Receptors / genetics
  • ErbB Receptors / metabolism*
  • Growth Hormone / pharmacology*
  • Humans
  • Receptors, Somatotropin / drug effects
  • Receptors, Somatotropin / genetics
  • Receptors, Somatotropin / metabolism*
  • Recombinant Fusion Proteins / drug effects
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism*

Substances

  • DNA Primers
  • Receptors, Somatotropin
  • Recombinant Fusion Proteins
  • Growth Hormone
  • ErbB Receptors