Abstract
A 60-kDa protein that undergoes rapid tyrosine phosphorylation in response to insulin and then binds phosphatidylinositol 3-kinase has been previously described in adipocytes and hepatoma cells. We have isolated this protein, referred to as pp60, from rat adipocytes, obtained the sequences of tryptic peptides, and cloned its cDNA. The predicted amino acid sequence of pp60 reveals that it contains an N-terminal pleckstrin homology domain, followed by a phosphotyrosine binding domain, followed by a group of likely tyrosine phosphorylation sites, four of which are in the YXXM motif that binds to the SH2 domains of phosphatidylinositol 3-kinase. The overall architecture of pp60 is thus the same as that of insulin receptor substrates 1 and 2 (IRS-1 and IRS-2), and furthermore both the pleckstrin homology and phosphotyrosine binding domains are highly homologous (about 50% identical amino acids) to these domains in both IRS-1 and IRS-2. Thus, pp60 is a new member of the IRS family, which we have designated IRS-3.
Highlights
The insulin receptor is a tyrosine kinase that is activated upon insulin binding
One protein of this type that hitherto has not been cloned is a 60-kDa protein described in rat adipocytes and rat hepatoma cells
The predicted amino acid sequence shows that pp60 is a new member of the IRS family
Summary
The insulin receptor is a tyrosine kinase that is activated upon insulin binding. Signaling from this receptor proceeds primarily by its tyrosine phosphorylation of substrate proteins, which act as docking proteins for one or more SH21 domain-containing proteins. 1 The abbreviations used are: SH2, Src homology 2; GST, glutathione S-transferase; GST-NSH2, GST fusion protein with N-terminal SH2 domain of phosphatidylinositol 3-kinase; HPLC, high performance liquid chromatography; IRS, insulin receptor substrate; PCR, polymerase chain reaction; PH, pleckstrin homology; PI, phosphatidylinositol; PTB, phosphotyrosine binding; RACE, rapid amplification of cDNA ends; bp, base pair(s); nt, nucleotide(s).
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