Re-engineering the target specificity of the insulin receptor by modification of a PTB domain binding site.
Shc and IRS-1 (and their relatives) are cytoplasmic docking proteins that possess phosphotyrosine-binding (PTB) domains, through which they bind specific activated receptor tyrosine kinases (RTK). The subsequent phosphorylation of Shc or IRS-1 creates binding sites for the SH2 domains of multiple signaling proteins, leading to the activation of intracellular biochemical pathways. The PTB domains of Shc and IRS-1 both recognize autophosphorylation sites in RTKs with the consensus sequence NPXpY, but show distinct abilities to bind stably to RTKs such as the TrkA nerve growth factor receptor and the insulin receptor. In vitro analysis has suggested that residues N-terminal to the NPXpY motif may determine the affinity with which phosphopeptide ligands are recognized by the Shc and IRS-1 PTB domains. Unlike IRS-1, the Shc PTB domain binds poorly to the insulin-receptor (IR) beta subunit in vitro, owing to its low affinity for the NPXpY autophosphorylation site at Tyr 960 of the IR. As a consequence, Shc does not bind stably to the activated IR in cells. We show that substitution of Ser 955, five residues N-terminal to the Tyr 960 autophosphorylation site (the -5 position), with Ile alters the target specificity of the IR such that it stably associates with Shc in insulin-stimulated cells. A triple substitution of the -5, -8 and -9 residues relative to Tyr 960 of the IR to the corresponding amino acids found in the Shc PTB domain binding site of TrkA results in even stronger binding of the IR to Shc in vivo. The variant IRs with enhanced ability to bind Shc showed an increased ability to activate the MAPK pathway in response to insulin stimulation. These results demonstrate that subtle differences in residues N-terminal to NPXpY autophosphorylation sites determine the ability of RTKs to bind specific PTB domain proteins in vivo, and thus modify the signaling properties of activated receptors.
van der Geer
P
P
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla 92093-0601, USA.
Wiley
S
S
Pawson
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D019556:COS Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D055504:Insulin Receptor Substrate Proteins; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D011233:Precipitin Tests; D011972:Receptor, Insulin / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D018909:src Homology Domains
D055504:Insulin Receptor Substrate Proteins; D010750:Phosphoproteins; D019000:Phosphotyrosine; D011972:Receptor, Insulin; D017871:Calcium-Calmodulin-Dependent Protein Kinases