Janes P W, Lackmann M, Church W B, Sanderson G M, Sutherland R L, Daly R J
Cancer Research Program, Garvan Institute of Medical Research, St. Vincent's Hospital, Sydney, New South Wales 2010, Australia.
J Biol Chem. 1997 Mar 28;272(13):8490-7. doi: 10.1074/jbc.272.13.8490.
The Src homology 2 (SH2) domain-containing protein Grb7 and the erbB2 receptor tyrosine kinase are overexpressed in a subset of human breast cancers. They also co-immunoprecipitate from cell lysates and associate directly in vitro. Whereas the Grb7 SH2 domain binds strongly to erbB2, the SH2 domain of Grb14, a protein closely related to Grb7, does not. We have investigated the preferred binding site of Grb7 within the erbB2 intracellular domain and the SH2 domain residues that determine the high affinity of Grb7 compared with Grb14 for this site. Phosphopeptide competition and site-directed mutagenesis revealed that Tyr-1139 of erbB2 is the major binding site for the Grb7 SH2 domain, indicating an overlap in binding specificity between the Grb7 and Grb2 SH2 domains. Substituting individual amino acids in the Grb14 SH2 domain with the corresponding residues from Grb7 demonstrated that a Gln to Leu change at the betaD6 position imparted high affinity erbB2 interaction, paralleled by a marked increase in affinity for the Tyr-1139 phosphopeptide. The reverse switch at the betaD6 position abrogated Grb7 binding to erbB2. This residue therefore represents an important determinant of SH2 domain specificity within the Grb7 family.
含Src同源2（SH2）结构域的蛋白Grb7和erbB2受体酪氨酸激酶在一部分人类乳腺癌中过表达。它们也能从细胞裂解物中共免疫沉淀，并且在体外直接结合。虽然Grb7的SH2结构域与erbB2强烈结合，但与Grb7密切相关的蛋白Grb14的SH2结构域则不能。我们研究了Grb7在erbB2细胞内结构域中的优先结合位点，以及与Grb14相比决定Grb7对该位点具有高亲和力的SH2结构域残基。磷酸肽竞争和定点诱变显示，erbB2的Tyr-1139是Grb7 SH2结构域的主要结合位点，这表明Grb7和Grb2的SH2结构域在结合特异性上存在重叠。用Grb7的相应残基替换Grb14 SH2结构域中的单个氨基酸表明，βD6位置的谷氨酰胺到亮氨酸的变化赋予了与erbB2的高亲和力相互作用，同时对Tyr-1139磷酸肽的亲和力也显著增加。βD6位置的反向转换消除了Grb7与erbB2的结合。因此，该残基代表了Grb7家族中SH2结构域特异性的一个重要决定因素。
