Identification of a heregulin binding site in HER3 extracellular domain

HER3 (also known as c-Erb-b3) is a type I receptor tyrosine kinase similar in sequence to the epidermal growth factor (EGF) receptor. The extracellula r segment of this transmembrane receptor contains four domains. Domains I a nd II are similar in sequence to domains III and IV, respectively, and doma ins II and IV are cysteine-rich. We show that the EGF-like domain of heregu lin (hrg) binds to domains I and II of HER3, in contrast to the EGF recepto r, for which prior studies have shown that a construct consisting of domain s III and portions of domain IV binds EGF. Next, we identified a putative h rg binding site by limited proteolysis of the recombinant extracellular dom ains of HER3 (HER3-ECDI-IV) in both the presence and absence of hrg. In the absence of hrg, HER3-ECDI-IV is cleaved after position Tyr(50), near the b eginning of domain I. Binding of hrg to HER3-ECDI-IV fully protects positio n Tyr(50) from proteolysis. To confirm that domain I contains a hrg binding site, we expressed domains I and II (HERS-ECDI-II) and find that it binds hrg with 68 nm affinity. These data suggest that domains I and II of HER3-E CDI-IV act as a functional unit in folding and binding of hrg. Thus, our bi ochemical findings reinforce the structural hypothesis of others that HER3- ECDI-IV is similar to the insulinlike growth factor-1 receptor (IGF-IR), as follows: 1) The protected cleavage site in HER3-ECDI-IV corresponds to a b inding footprint in domain I of IGF-1R; 2) HER3-ECDI-II binds hrg with a 68 nm dissociation constant, supporting the hypothesis that domain I is invol ved in ligand binding;, and 3) the large accessible surface area (1749 Angs trom) of domain L1 of IGF-1R that is buried by domain S1, as well as the pr esence of conserved contacts in this interface of type 1 RTKs, suggests tha t domains L1 and S1 of IGF-1R function as a unit as observed for HER3-ECDI- II. Our results are consistent with the propos that HER3 has a structure si milar to IGF-1R and binds ligand at a site in corresponding domains.