TY - JOUR
T1 - High-throughput selection of transmembrane sequences that enhance receptor tyrosine kinase activation
AU - He, Lijuan
AU - Hoffmann, Andrew R.
AU - Serrano, Christopher
AU - Hristova, Kalina
AU - Wimley, William C.
N1 - Funding Information:
This work was supported by NIH grants GM60000(WCW) and GM68619(KH).
PY - 2011/9/9
Y1 - 2011/9/9
N2 - Dimerization is a critical requirement for the activation of the intracellular kinase domains of receptor tyrosine kinases (RTKs). The single transmembrane (TM) helices of RTKs contribute to dimerization, but the details are not well understood. Work with TM helices in various model systems has revealed a small number of specific dimerization sequence motifs, and it has been suggested that RTK dimerization is modulated by such motifs. Yet questions remain about the universality of these sequence motifs for RTK dimerization and about how TM domain dimerization in model systems relates to RTK activation in mammalian membranes. To investigate these questions, we designed a 3888-member combinatorial peptide library based on the TM domain of Neu (ErbB2) as a model RTK. The library contains many closely related, Neu-like sequences, including thousands of sequences with known dimerization motifs. We used an SDS-PAGE-based screen to select peptides that dimerize better than the native Neu sequence, and we assayed the activation of chimeric Neu receptors in mammalian cells with TM sequences selected in the screen. Despite the very high abundance of known dimerization motifs in the library, only a very few dimerizing sequences were identified by SDS-PAGE. About half of those sequences activated the Neu kinase significantly more than did the wild-type TM sequence. This work furthers our knowledge about the requirements for membrane protein interactions and the requirements for RTK activation in cells.
AB - Dimerization is a critical requirement for the activation of the intracellular kinase domains of receptor tyrosine kinases (RTKs). The single transmembrane (TM) helices of RTKs contribute to dimerization, but the details are not well understood. Work with TM helices in various model systems has revealed a small number of specific dimerization sequence motifs, and it has been suggested that RTK dimerization is modulated by such motifs. Yet questions remain about the universality of these sequence motifs for RTK dimerization and about how TM domain dimerization in model systems relates to RTK activation in mammalian membranes. To investigate these questions, we designed a 3888-member combinatorial peptide library based on the TM domain of Neu (ErbB2) as a model RTK. The library contains many closely related, Neu-like sequences, including thousands of sequences with known dimerization motifs. We used an SDS-PAGE-based screen to select peptides that dimerize better than the native Neu sequence, and we assayed the activation of chimeric Neu receptors in mammalian cells with TM sequences selected in the screen. Despite the very high abundance of known dimerization motifs in the library, only a very few dimerizing sequences were identified by SDS-PAGE. About half of those sequences activated the Neu kinase significantly more than did the wild-type TM sequence. This work furthers our knowledge about the requirements for membrane protein interactions and the requirements for RTK activation in cells.
KW - RTK activation
KW - SDS PAGE
KW - high-throughput screening
KW - neu
KW - receptor tyrosine kinase
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U2 - 10.1016/j.jmb.2011.07.004
DO - 10.1016/j.jmb.2011.07.004
M3 - Article
C2 - 21767549
AN - SCOPUS:84860390410
SN - 0022-2836
VL - 412
SP - 43
EP - 54
JO - Journal of molecular biology
JF - Journal of molecular biology
IS - 1
ER -