Separation and characterization of three insulin receptor species that differ in subunit composition

Abstract

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 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. These results demonstrate that only the intact (.alpha..beta.)2 form of the insulin receptor has the ability to undergo insulin-stimulated kinase activity. The conversion of only one .beta. subunit to the .beta.'' subunit is enough to render the receptor incapable of ligand-induced autophosphorylation. However, the data also indicate that loss of one (or both) kinase domain(s) has no dramatic effect on the ability of the receptor''s binding site to interact with insulin.