Differential heat stress stability of epidermal growth factor receptor and erbB-2 receptor tyrosine kinase activities.
The epidermal growth factor (EGF) and erbB-2 receptors are structurally related membrane-bound tyrosine kinases. While these proteins exhibit close sequence homology, 50% overall and 80% in the tyrosine kinase domains, they respond very differently to heat stress. In NIH-3T3 or NR6 cells transfected with wild-type EGF-R and incubated at 37 degrees C or heat shocked at 46 degrees C, EGF binds to its receptor and stimulates receptor autophosphorylation to equivalent extents. At 46 degrees C, however, the basal tyrosine kinase activity of the wild-type erbB-2 receptor is rapidly lost. When cells containing chimeric receptors composed of the EGF-R extracellular domain and intracellular domain of erbB-2 were heat stressed, 125I-EGF bound to the receptors, but did not stimulate receptor autophosphorylation. The decline in EGF-stimulated chimeric erbB-2 receptor autophosphorylation is dependent on the length of heat shock, with nearly 100% of the kinase activity lost after 60 min at 46 degrees C. The loss of chimeric receptor erbB-2 kinase activity is not due to degradation of receptor protein, nor is it attributable to a specific transmembrane domain from either the EGF or erbB-2 receptors. Sensitivity of erbB-2 to heat stress is also not a result of denaturation of this receptor's carboxy-terminal domain. Insertion of the erbB-2 tyrosine kinase domain into the EGF-R confers heat stress sensitivity to the resultant chimeric receptor. Thus, although the EGF-R and erbB-2 kinase domains show a high degree of homology, the secondary/tertiary structures of these domains would seem to be stabilized in distinct manners.
Liu
S M
SM
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
Carpenter
G
G
D016475:3T3 Cells; D000818:Animals; D002460:Cell Line; D002678:Chimera; D004355:Drug Stability; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000032:analysis / Q000378:metabolism / Q000502:physiology*; D005347:Fibroblasts / Q000737:chemistry / Q000166:cytology / Q000648:ultrastructure; D006358:Hot Temperature / Q000009:adverse effects*; D015151:Immunoblotting; D007457:Iodine Radioisotopes; D051379:Mice; D010766:Phosphorylation; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000032:analysis / Q000378:metabolism / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000032:analysis / Q000378:metabolism / Q000502:physiology*; D018719:Erb-b2 Receptor Tyrosine Kinases; D013997:Time Factors; D014162:Transfection
D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D007457:Iodine Radioisotopes; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases