Structural studies of the detergent-solubilized and vesicle-reconstituted insulin receptor.
Insulin binding to the insulin receptor initiates a cascade of cellular events that are responsible for regulating cell metabolism, proliferation, and growth. We have investigated the structure of the purified, functionally active, human insulin receptor using negative stain and cryo-electron microscopy. Visualization of the detergent-solubilized and vesicle-reconstituted receptor shows the alpha(2)beta(2) heterotetrameric insulin receptor to be a three-armed pinwheel-like complex that exhibits considerable variability among individual receptors. The alpha-subunit of the receptor was labeled with an insulin analogue.streptavidin gold conjugate, which facilitated the identification of the receptor arm responsible for insulin binding. The gold label was localized to the tip of a single receptor arm of the three-armed complex. The beta-subunit of the insulin receptor was labeled with a maleimide-gold conjugate, which allowed orientation of the receptor complex in the membrane bilayer. The model derived from electron microscopic studies displays a "Y"-like morphology representing the predominant species identified in the reconstituted receptor images. The insulin receptor dimensions are approximately 12.2 nm by 20.0 nm, extending 9.7 nm above the membrane surface. The beta-subunit-containing arm is approximately 13.9 nm, and each alpha-subunit-containing arm is 8.6 nm in length. The model presented is the first description of the insulin receptor visualized in a fully hydrated state using cryo-electron microscopy.
Woldin
C N
CN
Department of Biophysics, Center for Advanced Biomedical Research, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Hing
F S
FS
Lee
J
J
Pilch
P F
PF
Shipley
G G
GG
D016475:3T3 Cells; D000818:Animals; D001710:Biotin; D004396:Coloring Agents; D020285:Cryoelectron Microscopy; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007150:Immunohistochemistry; D008301:Maleimides; D051379:Mice; D009942:Organometallic Compounds; D017434:Protein Structure, Tertiary; D011972:Receptor, Insulin / Q000737:chemistry* / Q000302:isolation & purification / Q000648:ultrastructure
D004396:Coloring Agents; D003902:Detergents; D008301:Maleimides; D009942:Organometallic Compounds; C043592:maleimide; C005460:uranyl acetate; D001710:Biotin; D011972:Receptor, Insulin