Hormone-triggered conformational changes within the insulin-receptor ectodomain: requirement for transmembrane anchors

Abstract

Interaction between two alphabeta half-receptors within the (alphabeta)(2) holoreceptor complex is required for insulin binding with high affinity and for insulin-triggered changes of size and shape. To understand the underlying structure-function relationship, two truncated receptor constructs have been characterized. Reduction in the Stokes radius and increase in the sedimentation coefficient, which are characteristic for wild-type receptors, were entirely lacking for the recombinant human insulin receptor (HIR) ectodomain (HIR-ED). Stokes radii of about 5.8 nm and sedimentation coefficients of 10.2 S were found for both insulin-bound and free HIR-EDs. However, attaching the membrane anchors to the ectodomain, as with the recombinant membrane-anchored ectodomain (HIR-MAED) construct, was sufficient to restore not only high-affinity hormone binding but also the marked insulin-inducible alterations in hydrodynamic properties. The Stokes radii of HIR-MAED complexes, as assessed by non-denaturing PAGE, decreased upon insulin binding from 9.5 nm to 7.9 nm. In parallel, the sedimentation coefficient was increased from 9.0 S to 9.8 S. CD and fluorescence spectroscopy of HIR-MAED revealed only minor insulin-induced changes in the secondary structure. Similarity with wild-type receptors has also been demonstrated by the differential insertion of insulin-bound and free HIR-MAED complexes into artificial bilayer membranes of Triton X-114. The results are consistent with a model of receptor function that ensures a global insulin-triggered reorientation of subdomains within the ectodomain moieties while the secondary structure is essentially retained. For the rearrangement of such subdomains, the transmembrane anchors confer essential structural constraints on the receptor ectodomain.

Identification of heat shock protein 5, calnexin and integral membrane protein 2B as Adam7‐interacting membrane proteins in mouse sperm

“…Triton X-114 partitions proteins into detergent and aqueous phases, depending on the conformation of proteins (Florke et al, 2001). We found that both L-Itm2b and S-Itm2b were found mainly in the detergent phase in both uncapacitated and capacitated sperm ( Fig.…”

Solution Structure of Ectodomains of the Insulin Receptor Family: The Ectodomain of the Type 1 Insulin-Like Growth Factor Receptor Displays Asymmetry of Ligand Binding Accompanied by Limited Conformational Change

“…Ultracentrifugation and gel-filtration studies 17 have shown that the detergentsolubilized form of wtIR undergoes significant compaction upon insulin binding but that such compaction does not occur in the ectodomain-only form of IR. 18 Corresponding studies with detergentsolubilized IGF-1R and IGF-1R ectodomain have not been reported. Attempts to investigate the conformational changes in wtIR upon ligand binding using dark-field scanning transmission electron microscopy have also been made, 19 but the models derived have been treated with some skepticism 20 and, in particular, show no correlation with the crystal structure of IR ectodomain homodimer.…”

Open conformers: the hidden face of MHC-I molecules

“…The first category results in the formation of homodimers (see earlier). The second category might include receptors such as CD8ab, that undergo physical changes in the CD8b chain following activation [66], and receptors that undergo conformational changes following binding to their physiological ligands, such as the insulin receptor [67]. The third type might include receptors that are expressed in metabolically active cells for the purpose of nutrient uptake and/or delivery of survival signals, such as the receptors for interleukin (IL)-2, IL-15 or iron-transferrin, all reported to cis-associate with classical and nonclassical MHC-I molecules [21,22,33,35,68,69].…”