DISTINCT ALPHA-SUBUNIT STRUCTURES OF HUMAN INSULIN RECEPTOR-A AND RECEPTOR-B VARIANTS DETERMINE DIFFERENCES IN TYROSINE KINASE-ACTIVITIES

Human insulin receptor isoforms (HIR-A and -B) differ in their a-subunit structures which result from alternatively spliced precursor mRNAs. This structural difference causes distinct binding affinities for insulin. To determine the impact of the structural difference on receptor signaling, we characterized the tyrosine kinase activity of HIR-A and HIR-B in vitro and determined the insulin stimulated beta-subunit phosphorylation and tyrosine kinase activation in the intact cell. When P-32 incorporation in beta-subunits of equal amounts of isolated HIR-A and HIR-B was measured, an increased P-32 incorporation in tyrosine residues of the beta-subunit of HIR-B (2.5-fold) compared to that of HIR-A was found after in vitro insulin stimulation. This was paralleled by an increased rate of phosphorylation (2.0-fold) of poly(GluNa,Tyr 4:1). In vitro analysis of K(m) values for ATP were similar for HIR-A (K(m) = 14.3-mu-M +/- 3.8) and HIR-B (K(m) = 20.2-mu-M +/- 8.6), whereas the V(max) of HIR-B was significantly increased (HIR-A V(max) = 5.5-mu-mol/60 min-mu-g-1 +/- 1.4, HIR-B V(max) = 42.5-mu-mol/60 min-mu-g-1 +/- 19.2). HPLC analysis of tryptic beta-subunit phosphopeptides revealed identical patterns, suggesting that the difference in kinase activities is not due to an alteration of the phosphorylation-activation cascade within the beta-subunit. However, when cleavage of the alpha-subunit by short-time trypsinization was used to activate the tyrosine kinase, the differences in P-32 incorporation between HIR-A and HIR-B were abolished. This suggests that the heterogeneity in the alpha-subunit structures of HIR-A and HIR-B determines the different kinase activities under basal and insulin-stimulated conditions. To assess whether this difference in kinase activities might be of physiological relevance, we studied receptor autophosphorylation in intact 293 cells transiently overexpressing HIR-A and HIR-B by immunoblotting of the beta-subunit with phosphotyrosine antibodies. There was no difference in the receptor autophosphorylation. However, when insulin receptors in intact rat-1 fibroblasts were stimulated with insulin and the receptor was subsequently isolated from the membrane to determine the phosphorylation of poly(GluNa,Tyr 4:1), HIR-B revealed a higher tyrosine kinase activity than HIR-A. These data suggest that HIR-B in solubilized form exists in a conformation that favors tyrosine kinase activation. The physiological significance of this finding, however, remains unknown.