title: Structural analysis and subunit interaction of insulin receptor from membranes of cultured embryonic chick heart cells.
abstract: In previous studies of cultured embryonic chick heart, insulin hyperpolarized cells and slowed their beat rate through occupancy of a high affinity receptor. In the present studies, we chemically characterize the native structure and subunit interactions of this insulin receptor. A stokes radius of 87 A and an apparent molecular mass of 350,000 daltons were found for membrane proteins specifically cross-linked to [125I] insulin by disuccinimidyl suberate. A primary subunit of 125,000 daltons in dithiothreitol or 115,000 daltons in its absence (alpha-subunit) was heavily cross-linked. A smaller subunit had a size of 90,000 daltons (beta-subunit). This beta-subunit was not readily labeled by [125I]insulin cross-linking, but insulin enhanced 32P incorporation from [gamma-32P]ATP, enabling its visualization. Subunit interaction could be studied, because alkaline conditions produced dissociation of the native 350,000-dalton receptor. This spontaneous dissociation was not a result of proteolysis and was prevented by acid conditions, oxidants, or N-ethylmaleimide. Using alkaline conditions followed by chemical reduction in two-way gels, we directly visualized the native complex of 350,000 daltons dissociating into combinations of subunits of apparent sizes of 290,000 (alpha 2 beta), 220,000 (alpha alpha), and 195,000 (alpha beta) daltons. Dithiothreitol produced combinations of subunits, which differed from alkaline dissociation. In alkaline conditions, the 290,000 (alpha 2 beta) and 220,000 (alpha alpha) dalton combinations predominated, whereas dithiothreitol produced 190,000 (alpha beta)-dalton proteins, which suggested that alpha-S-S-alpha disulfide bonds existed and were susceptible to chemical reductants, while alpha-S-S-beta disulfide bonds were more sensitive to alkaline lysis. We conclude from these observations that the native insulin receptor of embryonic chick heart cell exists on the sarcolemmal membrane as a relatively homogeneous tetramer of nonhomologous subunits in an alpha 2 beta 2 configuration. The alpha-subunits are the primary sites for insulin binding, and a beta-subunit is autophosphorylated. alpha-S-S-alpha and alpha-S-S-beta bonding exist, and these disulfide bonds have different sensitivities to chemical reducing agents and alkaline lysis.
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002642:Chick Embryo; D004591:Electrophoresis, Polyacrylamide Gel; D007328:Insulin / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation; D011972:Receptor, Insulin / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D007328:Insulin; D046911:Macromolecular Substances; C008147:insulin, iodo-; D000255:Adenosine Triphosphate; D011972:Receptor, Insulin