To examine the molecular basis of ligand-stimulated intramolecular beta-subunit autophosphorylation, hybrid receptors composed of wild-type and mutant insulin and insulin-like growth factor-1 (IGF-I) half-receptor precursors were characterized. Previous studies have demonstrated that assembly of the IGF-I wild-type half-receptor (alpha beta WT) with a kinase-defective half-receptor (alpha beta A/K) produced a substrate kinase-inactive holoreceptor in vitro [Treadway et al. (1991): Proc Natl Acad Sci USA 88:214-218]. To extend these studies, the vaccinia virus/bacteriophage T7 expression system was used to generate various hybrid receptor complexes in cultured cells. As was observed for hybrid receptors assembled in vitro, the wild-type/mutant hybrid receptors formed in situ were also incapable of phosphorylating several peptide substrates. However, ligand-stimulated beta-subunit autophosphorylation was still observed. To determine the molecular basis for this discrepancy, hybrid receptors were assembled from a truncated beta-subunit insulin half-receptor (alpha beta delta 43) and a kinase-defective half-receptor (alpha beta A/K). Under these conditions, insulin-stimulated autophosphorylation primarily occurred on the full-length kinase-inactive beta-subunit (alpha beta A/K) without significant labeling of the kinase-active truncated beta-subunit (alpha beta delta 43). A similar IGF-I hybrid receptor species was characterized, and the same pattern of autophosphorylation was observed in response to IGF-I. These data demonstrate that both insulin and IGF-I stimulate an intramolecular trans-autophosphorylation reaction between two adjacent beta-subunits within the holoreceptors.(ABSTRACT TRUNCATED AT 250 WORDS)