Alteration of epidermal growth factor receptor activity by mutation of its primary carboxyl-terminal site of tyrosine self-phosphorylation.
The epidermal growth factor (EGF) receptor, which exhibits intrinsic protein tyrosine kinase activity, undergoes a rapid, intramolecular self-phosphorylation reaction following EGF activation. The primary sites of tyrosine self-phosphorylation in vivo are located in the extreme carboxyl-terminal region of the molecule, principally Tyr-1173. To test the biological and biochemical consequences of this EGF receptor self-phosphorylation, we made the mutation Tyr----Phe-1173. Membranes containing the mutated receptor exhibited an ED50 for EGF activation of tyrosine kinase activity equivalent to control receptor at both high and low substrate levels, but exhibited reduced basal and EGF-stimulated tyrosine kinase activity at low, non-saturating substrate levels. The Tyr----Phe-1173 mutant possessed high affinity EGF binding and could still self-phosphorylate other tyrosine sites in an intramolecular fashion with a low Km for ATP (200 nM), suggesting that this alteration did not grossly change receptor structure. When EGF-dependent growth of Chinese hamster ovary cells expressing comparable levels of control or mutant EGF receptor was measured, the ability of the mutant receptor to mediate cell growth in response to EGF was reduced by approximately 50%, yet both receptors exhibited a similar affinity and ED50 for EGF. These results support the concept that this self-phosphorylation site can act as a competitive/alternate substrate for the EGF receptor, and that this region of the molecule is important in modulating its maximal biological activity.
Bertics
P J
PJ
Department of Physiological Chemistry, University of Wisconsin-Madison 53706.
Chen
W S
WS
Hubler
L
L
Lazar
C S
CS
Rosenfeld
M G
MG
Gill
G N
GN
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D066246:ErbB Receptors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005822:Genetic Vectors; D007700:Kinetics; D007739:L Cells / Q000378:metabolism; D051379:Mice; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids; D014158:Transcription, Genetic; D014443:Tyrosine
D014443:Tyrosine; D000255:Adenosine Triphosphate; D066246:ErbB Receptors