The insulin receptor tyrosine kinase domain in a chimaeric epidermal growth factor-insulin receptor generates Ca2+ signals through the PLC-gamma1 pathway.
The receptors for insulin (IR) and epidermal growth factor (EGFR) are members of the tyrosine kinase receptor (TKR) family. Despite homology of their cytosolic TK domains, both receptors induce different cellular responses. Tyrosine phosphorylation of insulin receptor substrate (IRS) molecules is a specific IR post-receptor response. The EGFR specifically activates phospholipase C-gamma1 (PLC-gamma1). Recruitment of substrate molecules with Src homology 2 (SH2) domains or phosphotyrosine binding (PTB) domains to phosphotyrosines in the receptor is one of the factors creating substrate specificity. In addition, it has been shown that the TK domains of the IR and EGFR show preferences to phosphorylate distinct peptides in vitro, suggesting additional mechanisms of substrate recognition. We have examined to what extent the substrate preference of the TK domain contributes to the specificity of the receptor in vivo. For this purpose we determined whether the IR TK domain, in situ, is able to tyrosine-phosphorylate substrates normally used by the EGFR. A chimaeric receptor, consisting of an EGFR in which the juxtamembrane and tyrosine kinase domains were exchanged by their IR counterparts, was expressed in CHO-09 cells lacking endogenous EGFR. This receptor was found to activate PLC-gamma1, indicating that the IR TK domain, in situ, is able to tyrosine phosphorylate substrates normally used by the EGFR. These findings suggest that the IR TK domain, in situ, has a low specificity for selection and phosphorylation of non-cognate substrates.
Telting
D
D
Department of Molecular Cell Biology, Leiden University Medical Centre, Sylvius Laboratory, Wassenaarseweg 72, 2333 AL, Leiden, The Netherlands.
Smeets
R L
RL
Willems
P H
PH
van der Zon
G C
GC
Frankhuizen
W S
WS
Maassen
J A
JA
D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000737:chemistry* / Q000494:pharmacology; D066246:ErbB Receptors / Q000096:biosynthesis / Q000737:chemistry; D007527:Isoenzymes / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D051966:Phospholipase C gamma; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011972:Receptor, Insulin / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*
D007527:Isoenzymes; D019000:Phosphotyrosine; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D011972:Receptor, Insulin; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D002118:Calcium