The C-terminal SH2 domain of p85 accounts for the high affinity and specificity of the binding of phosphatidylinositol 3-kinase to phosphorylated platelet-derived growth factor beta receptor.
Upon stimulation by its ligand, the platelet-derived growth factor (PDGF) receptor associates with the 85-kDa subunit of phosphatidylinositol (PI) 3-kinase. The 85-kDa protein (p85) contains two Src homology 2 (SH2) domains and one SH3 domain. To define the part of p85 that interacts with the PDGF receptor, a series of truncated p85 mutants was analyzed for association with immobilized PDGF receptor in vitro. We found that a fragment of p85 that contains a single Src homology domain, the C-terminal SH2 domain (SH2-C), was sufficient for directing the high-affinity interaction with the receptor. Half-maximal binding of SH2-C to the receptor was observed at an SH2-C concentration of 0.06 nM. SH2-C, like full-length p85, was able to distinguish between wild-type PDGF receptor and a mutant receptor lacking the PI 3-kinase binding site. An excess of SH2-C blocked binding of full-length p85 and PI 3-kinase to the receptor but did not interfere with the binding of two other SH2-containing proteins, phospholipase C-gamma and GTPase-activating protein. These results demonstrate that a region of p85 containing a single SH2 domain accounts both for the high affinity and specificity of binding of PI 3-kinase to the PDGF receptor.
Klippel
A
A
Howard Hughes Medical Institute, University of California, San Francisco 94143-0724.
Escobedo
J A
JA
Fantl
W J
WJ
Williams
L T
LT
D016475:3T3 Cells; D000818:Animals; D001665:Binding Sites; D004252:DNA Mutational Analysis; D020690:GTPase-Activating Proteins; D051379:Mice; D019869:Phosphatidylinositol 3-Kinases; D010770:Phosphotransferases / Q000737:chemistry / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000378:metabolism*; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D017479:Receptors, Platelet-Derived Growth Factor; D013329:Structure-Activity Relationship; D010738:Type C Phospholipases / Q000378:metabolism
D020690:GTPase-Activating Proteins; D010982:Platelet-Derived Growth Factor; D011506:Proteins; D010770:Phosphotransferases; D011494:Protein Kinases; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; D017479:Receptors, Platelet-Derived Growth Factor; D010738:Type C Phospholipases