Re-engineering the target specificity of the insulin receptor by modification of a PTB domain binding site

Citation
P. Van Der Geer et al., Re-engineering the target specificity of the insulin receptor by modification of a PTB domain binding site, ONCOGENE, 18(20), 1999, pp. 3071-3075
Citations number
22
Categorie Soggetti
Onconogenesis & Cancer Research
Journal title
ONCOGENE
ISSN journal
09509232 → ACNP
Volume
18
Issue
20
Year of publication
1999
Pages
3071 - 3075
Database
ISI
SICI code
0950-9232(19990520)18:20<3071:RTTSOT>2.0.ZU;2-9
Abstract
Shc and IRS-1 (and their relatives) are cytoplasmic docking proteins that p ossess phosphotyrosine-binding (PTB) domains, through which they bind speci fic activated receptor tyrosine kinases (RTK), The subsequent phosphorylati on of Shc or IRS-1 creates binding sites for the SH2 domains of multiple si gnaling proteins, leading to the activation of intracellular biochemical pa thways, The PTB domains of Shc and IRS-1 both recognize autophosphorylation sites in RTKs with the consensus sequence NPXpY, but show distinct abiliti es 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-term inal to the NPXpY motif may determine the affinity with which phosphopeptid e ligands are recognized by the Shc and IRS-1 PTB domains, Unlike IRS-1, th e 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 activa ted IR in cells. We show that substitution of Ser 955, five residues N-term inal to the Tyr 960 autophosphorylation site (the -5 position), with Ile al ters the target specificity of the IR such that it stably associates with S hc 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 fou nd in the Shc PTB domain binding site of TrkA results in even stronger bind ing of the IR to Shc in vivo, The variant IRs with enhanced ability to bind Shc showed an increased ability Ito activate the MAPK pathway in response to insulin stimulation. These results demonstrate that subtle differences i n residues N-terminal to NPXpY autophosphorylation sites determine the abil ity of RTKs tea bind specific PTB domain proteins in vivo, and thus modify the signaling properties of activated receptors.