α-Helical element at the hormone-binding surface of the insulin receptor functions as a signaling element to activate its tyrosine kinase.
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 dimer-related 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 photo-probes 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.
Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106, USA. jonathan.whittaker@case.edu
Jonathan
J
Whittaker
Linda J
LJ
Whittaker
Charles T
CT
Roberts
Nelson B
NB
Phillips
Faramarz
F
Ismail-Beigi
Michael C
MC
Lawrence
Michael A
MA
Weiss
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D001407:Bacillus / Q000378:metabolism; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D018360:Crystallography, X-Ray / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism; D006728:Hormones / Q000378:metabolism; D008954:Models, Biological; D008968:Molecular Conformation; D016296:Mutagenesis; D009154:Mutation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry*; D011972:Receptor, Insulin / Q000378:metabolism*; D015398:Signal Transduction; D014443:Tyrosine / Q000737:chemistry
D003432:Cross-Linking Reagents; D006728:Hormones; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D000604:Amino Acyl-tRNA Synthetases