Structural organization of the human insulin receptor ectodomain.
To provide an experimental system amenable to a detailed biochemical and structural investigation of the extracellular (ligand binding) domain of the insulin receptor, we developed a mammalian heterologous cell expression system from which tens of milligrams of the soluble secreted ectodomain (the IR921 protein) can be routinely purified using methods that do not require harsh elution conditions. The purified IR921 protein has a Stokes radius of 6.8 nm and a sedimentation coefficient of 9.8 S, from which we calculate a hydro-dynamic mass of 281 kDa. Electron microscopic images, using both rotary shadowing and negative staining techniques, demonstrate a characteristic substructure for the IR921 protein consisting of two elongated arms, with a globular domain at each end, connected to each other at a point somewhat off-center to form a Y structure. Analysis using circular dichroism and fluorescence spectroscopy illustrate that insulin binding results in conformational changes in the ectodomain. Furthermore, fluorescence anisotropy decay data reveal segmental mobility within the IR921 protein that is successively frozen as a result of insulin binding, in contrast to results obtained in a previous study of the epidermal growth factor receptor ectodomain. This result suggests a divergence in hormone-induced signaling mechanisms used by the insulin and epidermal growth factor receptors.
Schaefer
E M
EM
Institute of Biosciences and Technology, Texas A & M University, Houston 77030.
Erickson
H P
HP
Federwisch
M
M
Wollmer
A
A
Ellis
L
L
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D015816:Cell Adhesion Molecules, Neuronal / Q000737:chemistry; D002942:Circular Dichroism; D006224:Cricetinae; D016326:Extracellular Matrix Proteins / Q000737:chemistry; D013006:Growth Hormone / Q000737:chemistry; D006801:Humans; D008433:Mathematics; D008854:Microscopy, Electron; D008961:Models, Structural; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011972:Receptor, Insulin / Q000737:chemistry* / Q000302:isolation & purification / Q000648:ultrastructure; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000648:ultrastructure; D012091:Repetitive Sequences, Nucleic Acid; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D019063:Tenascin; D014162:Transfection
D015816:Cell Adhesion Molecules, Neuronal; D016326:Extracellular Matrix Proteins; D011994:Recombinant Proteins; D019063:Tenascin; D013006:Growth Hormone; D011972:Receptor, Insulin