TI  - Characterization of an interaction between  <prot>insulin receptor substrate 1</prot> 
      and the  <prot>insulin receptor</prot>  by using the two-hybrid system.
PG  - 6433-42
AB  -  <prot>Insulin receptor substrate 1</prot> (<prot>IRS-1</prot>)  is a major substrate of the  <prot>insulin
      receptor</prot>  and has been implicated in <prot>insulin</prot> signaling. Although <prot>IRS-1</prot> is
      thought to interact with the <prot><prot>insulin</prot> receptor</prot>, the nature of the
      interaction has not been defined. In this study, we used the two-hybrid
      assay of protein-protein interaction in the yeast Saccharomyces cerevisiae
      to study the interaction between human <prot>IRS-1</prot> and the <prot><prot>insulin</prot> receptor</prot>. We
      demonstrate that  <prot>IRS-1</prot>  forms a specific complex with the cytoplasmic
      domain of the  <prot>insulin receptor</prot>  when both are expressed as hybrid proteins
      in yeast cells. We show that the interaction is strictly dependent upon
      receptor tyrosine kinase activity, since <prot>IRS-1</prot> shows no interaction with a
      kinase-inactive receptor hybrid containing a mutated ATP-binding site.
      Furthermore, mutation of receptor tyrosine 960 to phenylalanine eliminates
      <prot>IRS-1</prot> interaction in the two-hybrid assay. These data suggest that the
      interaction between <prot>IRS-1</prot>  and the  receptor is direct and provide evidence
      that the juxtamembrane domain of the receptor is involved. Furthermore, we
      show that a 356-amino-acid region encompassed by amino acids 160 through
      516 of <prot>IRS-1</prot> is sufficient for interaction with the receptor in the
      two-hybrid assay. Lastly, in agreement with our findings for yeast cells,
      we show that the <prot>insulin receptor</prot> is unable to phosphorylate an <prot>IRS-1</prot>
      protein containing a deletion of amino acids 45 to 516 when expressed in
      COS cells. The two-hybrid assay should provide a facile means by which to
      pursue a detailed understanding of this interaction.
AD  - Department of Physiology, University of Maryland School of Medicine,
