Structural Studies of the Detergent-solubilized and Vesicle-reconstituted Insulin Receptor

The insulin receptor (IR) 1 belongs to the superfamily of transmembrane receptor tyrosine kinases (TKs) (reviewed in Ref. 1). In contrast to other family members that are monomeric in their structure, IR and its homologue, insulin-like growth factor I receptor (IGF-1R), are intrinsic disulfide-linked dimers of heterodimeric disulfide-linked proteins of the form (␣␤) 2 . The 135-kDa ␣ subunit of IR is extracellular, whereas the 95-kDa ␤ subunit contains an extracellular portion, a single transmembrane sequence, and an intracellular TK domain. Fig. 1a depicts the major structural features of the ␣␤ dimer (see review by Tavaré and Siddle (2)). Ligandspecific binding to the ␣ subunits activates the TK, initiating a signal cascade that results in numerous cellular responses. Our understanding of the mechanics of this signal transduction process has been hampered by the unavailability of an atomic structure of the whole IR protein. However, the quaternary structure of the isolated complex of biologically active IR and insulin was recently solved by three-dimensional reconstruction from low dose scanning transmission electron micrographs (STEM) (3) (Fig. 1, b1-b3). Atomic structures of subdomains of IR or of highly analogous proteins were fitted into the complex (e.g. Fig. 1, b4), creating the only available atomic structural model of IR. The model reveals structural details of the interaction of insulin with the receptor that lead to the activation of the intracellular TK (4). Here we review previous biochemical observations on IR binding of natural and modified insulins and of insulin-like growth factor 1 (IGF-1) against this atomic structural model, and in the light of recent structural data on the unbound receptor, we discuss the mechanics of a model of receptor activation arising from insulin binding. Three-dimensional STEMAlthough crystallography remains the technique of choice for atomic structure determination of large proteins, considerable advances in electron microscopy (EM) and in image processing have been made for three-dimensional structure determination of proteins that are refractory to crystallization. For a growing number of proteins, including the plant light-harvesting complex (5), a human water channel (6), and aquaporin-1 (7), three-dimensional structural information has been obtained at resolutions of several Angstrom units, revealing ␣-helices and other structural details.In STEM, image acquisition is digital, as a 3-Å small electron beam probe scans across the specimen at low temperature. Signal intensity is directly proportional to the molecular mass of each molecule. Moreover, STEM in dark field mode can readily visualize clusters of heavy atoms on specifically marked biological molecules (3,8). STEM images have been used to reconstruct three-dimensional structures of several proteins at resolutions of 12-20 Å, such as SRP54, the Klenow fragment of DNA polymerase I, and IR (3, 9 -12).The STEM IR reconstruction at 20 Å was docked with atomic structures of insulin and of subdomains of IR or hi...

Section: Minireview: Three-dimensional Structural Interactions 27331mentioning

confidence: 82%

“…3d). Moreover, this structure, together with the insulinbound three-dimensional structure, makes it possible to reconcile (45)(46)(47)(48). Sixth column, modeled structures based on Fig.…”

Section: The Mechanics Of Transmembrane Signalingmentioning

confidence: 99%

Section: Minireview: Three-dimensional Structural Interactions 27331mentioning

confidence: 99%

See 1 more Smart Citation

Three-dimensional Structural Interactions of Insulin and Its Receptor

Yip

Ottensmeyer

2003

“…Our structural studies of the covalent disulfide-linked ␣ 2 ␤ 2 heterodimeric insulin receptor do indeed reveal a Y-shaped receptor (Ref. 40; see also Ref. 41).…”

Section: Figmentioning

confidence: 99%

Vesicle-reconstituted Low Density Lipoprotein Receptor

Jeon¹,

Shipley

2000

Self Cite

The low density lipoprotein (LDL) receptor is a key protein for maintaining cellular cholesterol homeostasis by binding cholesterol-rich lipoproteins through their apoB and apoE apoproteins. The LDL receptor is a transmembrane glycoprotein of M r ϳ115 kDa; based on its primary sequence, five distinct structural domains have been identified (Yamamoto, T., Davis, C. G., Brown, M. S., Schneider, W. J., Casey, M. L., Goldstein, J. L., and Russell, D. W. (1984) Cell 39, 27-38). As a first step toward providing a structural description of the intact LDL receptor, the receptor has been purified from bovine adrenal cortices, reconstituted into unilamellar egg yolk phosphatidylcholine vesicles, and imaged using cryoelectron microscopy (cryoEM). CryoEM has the advantage of providing images of the reconstituted LDL receptor in its frozen, fully hydrated state. LDL receptor molecules were visualized as elongated, stick-like projections from the vesicle surface with maximum dimensions ϳ120-Å length by ϳ45-Å width. In some of the images, a short arm (or arms) was visible at the distal end of the stick-like projections. The LDL receptor was labeled via accessible free cysteine residues, probably including that corresponding to Cys-431 of the known fulllength sequence of the human LDL receptor. The accessible cysteine was demonstrated using a maleimide-biotin⅐streptavidin conjugate and confirmed by labeling with monomaleimido-Nanogold. Images obtained by cryoEM showed that the extracellular stick-like domain of the reconstituted LDL receptor was labeled by Nanogold. This combined cryoEM-Nanogold labeling study has provided the first low resolution structural images of the reconstituted, full-length bovine LDL receptor. Low density lipoprotein (LDL)1 is the most abundant cholesterol-containing lipoprotein in human plasma (1). Since an elevated level of blood cholesterol is a major risk factor in developing atherosclerosis, the LDL receptor, which is responsible for the uptake of LDL into cells, has been studied extensively, notably by Brown and Goldstein (2). The LDL receptor is located in coated pit regions on the cell surface; it binds cholesterol-rich lipoproteins (LDL and ␤-VLDL) via receptor binding sub-domains of their apoB and apoE proteins, and the receptor-ligand complex is internalized by endocytosis. Acidification of the internal compartment of the endocytic vesicle leads to dissociation of the LDL receptor-LDL complex; the LDL receptor is recycled to the cell surface, and LDL is delivered to the lysosome. Release of cholesterol triggers a variety of intracellular metabolic and regulatory events, notably the regulation of cholesterol synthesis and expression of the LDL receptor and other genes (1-4). It should be stated that these studies of the LDL receptor were critical not only in providing a rationale for cholesterol homeostasis but also for laying the foundations for our understanding of intracellular trafficking mechanisms.The amino acid sequence of the LDL receptor has been reported for several species includin...

“…These studies have suggested either extended Tor Y-shaped receptor conformations (11,42,43) or more globular conformations (44 -46) but no clear consensus has emerged. The high affinity obtained with mIR.Fn0/Ex10 suggests that the first four domains, L1/Cys/L2/Fn0 and the Ex10 domain are in close proximity in the IR structure and that these most likely are the only domains interacting with the insulin molecule.…”

Section: Table I Insulin Affinity Of Recombinant Receptors In Variousmentioning

confidence: 99%

Dimeric Fragment of the Insulin Receptor α-Subunit Binds Insulin with Full Holoreceptor Affinity

Brandt

Andersen

Kristensen

2001

The insulin receptor (IR) is a dimeric receptor, and its activation is thought to involve cross-linking between monomers initiated by binding of a single insulin molecule to separate epitopes on each monomer. We have previously shown that a minimized insulin receptor consisting of the first three domains of the human IR fused to 16 amino acids from the C-terminal of the ␣-subunit was monomeric and bound insulin with nanomolar affinity (Kristensen, C., Wiberg, F. C., Schä ffer, L., and Andersen, A. S. (1998) J. Biol. Chem. 273, 17780 -17786). To investigate the insulin binding properties of dimerized ␣-subunits, we have reintroduced the domains containing ␣-␣ disulfide bonds into this minireceptor. When inserting either the first fibronectin type III domain or the full-length sequence of exon 10, the receptor fragments were predominantly secreted as disulfide-linked dimers that both had nanomolar affinity for insulin, similar to the affinity found for the minireceptor. However, when both these domains were included we obtained a soluble dimeric receptor that bound insulin with 1000-fold higher affinity (4 -8 pM) similar to what was obtained for the solubilized holoreceptor (14 -24 pM). Moreover, dissociation of labeled insulin from this receptor was accelerated in the presence of unlabeled insulin, demonstrating another characteristic feature of the holoreceptor. This is the first direct demonstration showing that the ␣-subunit of IR contains all the epitopes required for binding insulin with full holoreceptor affinity.