Separation and characterization of three insulin receptor species that differ in subunit composition.
Partially purified human placental insulin receptor preparations give rise to three distinct insulin-binding peaks when eluted from a Mono Q high-performance liquid chromatography anion-exchange column. We analyzed the basis for this phenomenon by affinity cross-linking of insulin to each peak, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We find that the three insulin-binding peaks represent different molecular weight complexes with the following subunit composition: (alpha beta)2, (alpha beta)(alpha beta'), and (alpha beta')2, where beta' represents a proteolytically derived fragment of the beta subunit. This analysis of subunit composition was confirmed by silver staining of affinity-purified insulin receptor following resolution of the forms on a Mono Q column as described previously. We have characterized the three isolated insulin receptor forms with regard to ligand binding by LIGAND and Scatchard analysis. We also measured insulin-stimulatable autophosphorylation and exogenous kinase activity directed toward poly(Glu/Tyr) (4:1). The three forms of the insulin receptor exhibit similar KD's for insulin binding to the high- and low-affinity sites. The (alpha beta)2 and (alpha beta)(alpha beta') forms of the insulin receptor display superimposable curvilinear Scatchard plots. In contrast, only the intact holoreceptor (alpha beta)2 form demonstrates insulin-stimulatable autophosphorylation and exogenous kinase activity. The (alpha beta)(alpha beta') form has reduced basal kinase activity which was not increased by prior incubation with insulin. The (alpha beta')2 form lacks a kinase domain and consequently demonstrated no kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)
D000345:Affinity Labels; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D006801:Humans; D066298:In Vitro Techniques; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010766:Phosphorylation; D010920:Placenta / Q000032:analysis; D011494:Protein Kinases / Q000378:metabolism; D011972:Receptor, Insulin / Q000145:classification / Q000302:isolation & purification* / Q000502:physiology; D013329:Structure-Activity Relationship
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D007328:Insulin; D046911:Macromolecular Substances; D011494:Protein Kinases; D011972:Receptor, Insulin