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
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.