Inhibition of hGrb10 binding to the insulin receptor by functional domain-mediated oligomerization.
hGrb10 is a newly identified Src homology 2 (SH2) and pleckstrin homology (PH) domain-containing protein that binds to autophosphorylated receptor tyrosine kinases, including the insulin and insulin-like growth factor receptors. To identify potential downstream proteins that interact with hGrb10, we screened a yeast two-hybrid cDNA library using the full-length hGrb10gamma as bait. A fragment of hGrb10, which included the IPS (insert between the PH and SH2 domain) and the SH2 domains, was found to bind with high affinity to the full-length protein. The interaction between the IPS/SH2 domain and the full-length hGrb10 was further confirmed by in vitro glutathione S-transferase fusion protein binding studies. Gel filtration assays showed that hGrb10 underwent tetramerization in mammalian cells. The interaction involved at least two functional domains, the IPS/SH2 region and the PH domain, both of which interacted with the NH2-terminal amino acid sequence of hGrb10gamma (hGrb10gamma DeltaC, residues 4-414). Competition studies showed that hGrb10gamma DeltaC inhibited the binding of hGrb10 to the tyrosine-phosphorylated insulin receptor, suggesting that this region may play a regulatory role in hGrb10/insulin receptor interaction. We present a model for hGrb10 tetramerization and its potential role in receptor tyrosine kinase signal transduction.
Dong
L Q
LQ
Department of Pharmacology, The University of Texas Health Science Center, San Antonio, Texas 78284-7764, USA.
Porter
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S
Hu
D
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Liu
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D000818:Animals; D001704:Biopolymers; D016466:CHO Cells; D006224:Cricetinae; D051383:GRB10 Adaptor Protein; D006801:Humans; D011485:Protein Binding; D011506:Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011972:Receptor, Insulin / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D015398:Signal Transduction; D018909:src Homology Domains
D001704:Biopolymers; D011506:Proteins; D011994:Recombinant Proteins; D051383:GRB10 Adaptor Protein; D011972:Receptor, Insulin