TY - JOUR
T1 - α-Helical element at the hormone-binding surface of the insulin receptor functions as a signaling element to activate its tyrosine kinase
AU - Whittaker, Jonathan
AU - Whittaker, Linda J.
AU - Roberts, Charles T.
AU - Phillips, Nelson B.
AU - Ismail-Beigi, Faramarz
AU - Lawrence, Michael C.
AU - Weiss, Michael A.
PY - 2012/7/10
Y1 - 2012/7/10
N2 - The primary hormone-binding surface of the insulin receptor spans one face of the N-terminal β-helix of the α-subunit (the L1 domain) and an α-helix in its C-terminal segment (αCT). Crystallographic analysis of the free ectodomain has defined a contiguous dimerrelated motif in which the αCT α-helix packs against L1 β-strands 2 and 3. To relate structure to function, we exploited expanded genetic-code technology to insert photo-activatable probes at key sites in L1 and αCT. The pattern of αCT-mediated photo-cross-linking within the free and bound receptor is in accord with the crystal structure and prior mutagenesis. Surprisingly, L1 photoprobes in β-strands 2 and 3, predicted to be shielded by αCT, efficiently cross-link to insulin. Furthermore, anomalous mutations were identified on neighboring surfaces of αCT and insulin that impair hormone-dependent activation of the intracellular receptor tyrosine kinase (contained within the transmembrane β-subunit) disproportionately to their effects on insulin binding. Taken together, these results suggest that αCT, in addition to its hormone-recognition role, provides a signaling element in the mechanism of receptor activation.
AB - The primary hormone-binding surface of the insulin receptor spans one face of the N-terminal β-helix of the α-subunit (the L1 domain) and an α-helix in its C-terminal segment (αCT). Crystallographic analysis of the free ectodomain has defined a contiguous dimerrelated motif in which the αCT α-helix packs against L1 β-strands 2 and 3. To relate structure to function, we exploited expanded genetic-code technology to insert photo-activatable probes at key sites in L1 and αCT. The pattern of αCT-mediated photo-cross-linking within the free and bound receptor is in accord with the crystal structure and prior mutagenesis. Surprisingly, L1 photoprobes in β-strands 2 and 3, predicted to be shielded by αCT, efficiently cross-link to insulin. Furthermore, anomalous mutations were identified on neighboring surfaces of αCT and insulin that impair hormone-dependent activation of the intracellular receptor tyrosine kinase (contained within the transmembrane β-subunit) disproportionately to their effects on insulin binding. Taken together, these results suggest that αCT, in addition to its hormone-recognition role, provides a signaling element in the mechanism of receptor activation.
KW - Affinity
KW - Alanine scanning
KW - Nonstandard mutagenesis
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UR - http://www.scopus.com/inward/citedby.url?scp=84863884698&partnerID=8YFLogxK
U2 - 10.1073/pnas.1205681109
DO - 10.1073/pnas.1205681109
M3 - Article
C2 - 22736795
AN - SCOPUS:84863884698
SN - 0027-8424
VL - 109
SP - 11166
EP - 11171
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 28
ER -