TY - JOUR
T1 - Identification of a Heregulin Binding Site in HER3 Extracellular Domain
AU - Singer, Elizabeth
AU - Landgraf, Ralf
AU - Horan, Tom
AU - Slamon, Dennis
AU - Eisenberg, David
N1 - Copyright:
Copyright 2008 Elsevier B.V., All rights reserved.
PY - 2001/11/23
Y1 - 2001/11/23
N2 - 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 extracellular segment of this transmembrane receptor contains four domains. Domains I and II are similar in sequence to domains III and IV, respectively, and domains II and IV are cysteine-rich. We show that the EGF-like domain of heregulin (hrg) binds to domains I and II of HER3, in contrast to the EGF receptor, for which prior studies have shown that a construct consisting of domains III and portions of domain IV binds EGF. Next, we identified a putative hrg binding site by limited proteolysis of the recombinant extracellular domains of HER3 (HER3-ECD I-V) in both the presence and absence of hrg. In the absence of hrg, HER3-ECDI-V is cleaved after position Tyr50, near the beginning of domain I. Binding of hrg to HER3-ECDI-V fully protects position Tyr50 from proteolysis. To confirm that domain I contains a hrg binding site, we expressed domains I and II (HER3-ECDI-II) and find that it binds hrg with 68 nM affinity. These data suggest that domains I and II of HER3-ECDI-IV act as a functional unit in folding and binding of hrg. Thus, our biochemical findings reinforce the structural hypothesis of others that HER3-ECDI-IV is similar to the insulin-like growth factor-1 receptor (IGF-1R), as follows: 1) The protected cleavage site in HER3-ECDI-IV corresponds to a binding 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 involved in ligand binding; and 3) the large accessible surface area (1749 Å) of domain L1 of IGF-1R that is buried by domain S1, as well as the presence of conserved contacts in this interface of type 1 RTKs, suggests that domains L1 and S1 of IGF-1R function as a unit as observed for HER3-ECDI-II. Our results are consistent with the proposal that HER3 has a structure similar to IGF-1R and binds ligand at a site in corresponding domains.
AB - 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 extracellular segment of this transmembrane receptor contains four domains. Domains I and II are similar in sequence to domains III and IV, respectively, and domains II and IV are cysteine-rich. We show that the EGF-like domain of heregulin (hrg) binds to domains I and II of HER3, in contrast to the EGF receptor, for which prior studies have shown that a construct consisting of domains III and portions of domain IV binds EGF. Next, we identified a putative hrg binding site by limited proteolysis of the recombinant extracellular domains of HER3 (HER3-ECD I-V) in both the presence and absence of hrg. In the absence of hrg, HER3-ECDI-V is cleaved after position Tyr50, near the beginning of domain I. Binding of hrg to HER3-ECDI-V fully protects position Tyr50 from proteolysis. To confirm that domain I contains a hrg binding site, we expressed domains I and II (HER3-ECDI-II) and find that it binds hrg with 68 nM affinity. These data suggest that domains I and II of HER3-ECDI-IV act as a functional unit in folding and binding of hrg. Thus, our biochemical findings reinforce the structural hypothesis of others that HER3-ECDI-IV is similar to the insulin-like growth factor-1 receptor (IGF-1R), as follows: 1) The protected cleavage site in HER3-ECDI-IV corresponds to a binding 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 involved in ligand binding; and 3) the large accessible surface area (1749 Å) of domain L1 of IGF-1R that is buried by domain S1, as well as the presence of conserved contacts in this interface of type 1 RTKs, suggests that domains L1 and S1 of IGF-1R function as a unit as observed for HER3-ECDI-II. Our results are consistent with the proposal that HER3 has a structure similar to IGF-1R and binds ligand at a site in corresponding domains.
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U2 - 10.1074/jbc.M105428200
DO - 10.1074/jbc.M105428200
M3 - Article
C2 - 11555649
AN - SCOPUS:0035941258
VL - 276
SP - 44266
EP - 44274
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 47
ER -