Biochemistry zyxwvut 1994,33, zyxwvu 9 143-9 151 9143 Deletion of 343 Amino Acids from the Carboxyl Terminus of the P-Subunit of the Insulin Receptor Inhibits Insulin Signaling Carol Renfrew Haft and Simeon I. Taylor' Diabetes Branch, National Institute zyxwvuts of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892 Received February zyxwvuts 3, 1994; Revised Manuscript Received April 28, 1994' ABSTRACT: Naturally occurring mutations in the insulin receptor gene that impair the receptor tyrosine kinase activity cause insulin resistance in uiuo in a dominant fashion. Previously, two unrelated families have been described that express an insulin receptor with a truncation due to a premature chain termination at codon 1000 (AlOOO), thereby deleting 343 amino acids from the carboxyl terminus of the @-subunit. While clinical findings suggest that the truncated receptor does not mediate insulin action in uiuo, a recent study suggested that a similarly truncated receptor enhanced insulin sensitivity in transfected cells by augmenting the signaling by endogenous receptors [Sasaoka, T., Takata, Y., Kusari, J., Anderson, zy C. M., Langlois, W. J., zyxwvuts & Olefsky, J. M. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 437943831, To investigate these paradoxical data, we studied the structure and function of A1 000 truncated insulin receptors when expressed in NIH-3T3 cells. We found that, despite the deletion of most of the tyrosine kinase domain and all of the C-terminal domain of the P-subunit of the insulin receptor, the AlOOO mutant receptors were processed normally and were transported to the plasma membrane where they bind insulin with high affinity. Following ligand addition, the truncated receptors are degraded with a normal half-life. However, they fail to undergo insulin-stimulated internalization, do not regulate the phosphorylation of insulin receptor substrate 1, and are unable to mediate an insulin-stimulated increase in DNA synthesis and c-jun and c-fos expression. These results demonstrate that the AlOOO truncated receptors, expressed in our in vitro model system, faithfully mirror the in zyxwvutsrq uiuo findings that this mutation causes insulin resistance. The insulin receptor is a heterotetrameric membrane protein that belongs to the family of ligand-activated protein tyrosine kinases (Ullrich et al., 1985; Ebina et al., 1985; Hanks et al., 1988). Insulin binding to the a-subunit of its receptor stimulates autophosphorylation of multiple tyrosine residues (Kasuga et al., 1982a,b;Tornqvist etal., 1987,1988;Tornqvist & Avruch, 1988) and activates the tyrosine kinase activity of the receptor @-subunit (Herrera & Rosen, 1986;Rosen, 1987). Following these events a variety of cellular substrates are phosphorylated, including insulin receptor substrate 1 (IRS- l),' and numerous biologic effects are initiated (Chou et al., 1987; Ellis et al., 1986; Sun et al., 1991; Backer et al., 1993). At present, the structural features of the insulin receptor moleculethat mediate its interactions with cellular substrates, such as Shc and IRS-1, remain poorly defined. Several types of mutations in the insulin receptor gene have been identified in patients with inherited syndromesof extreme insulin resistance (Tayloret al., 1992). Someoftheseseverely insulin-resistant patients are compound heterozygotes, pos- sessing two different mutant alleles of the insulin receptor gene (Kadowaki et al., 1988, 1990a; Barbetti et al., 1992; Kusari et al., 1991). Others are simple heterozygotes, possessing one normal allele and one mutant allele. Many of these simple heterozygotes possess one insulin receptor allele that is defective in its tyrosine kinase domain. Futhermore, patients who are heterozygous for a kinase defective receptor appear to be more severely insulin-resistant than patients who are heterozygous for a null allele (Moller & Flier, 1988; * Address correspondenceto this author at the National Institutes of e Abstract published in Aduance ACS Abstracts, July 15, 1994. Abbreviations: IRS-1, insulin receptor substrate 1; Shc, SH2- containing sequence;PBS, phosphate-bufferedsaline;BSA, bovine serum albumin; ECL, enhanced chemiluminescence. Health, Building 10, Room 89-239, Bethesda, MD 20892. Odawara et al., 1989; Cama et al., 1991, 1993). At present, the mechanism underlying this dominant negative effect is unclear. It has been suggested that mutant receptors may form hybrids with wild-type receptors leading to inactive oligomers (Frattali, et al., 1992; Chin et al., 1991). It is also possible that mutant receptors compete with wild-type recep- tors for a limited number of cellular substrate molecules involved in the insulin signal transduction cascade (Maegawa et al., 1988; Levy-Toledano et al., 1994). Previously, two unrelated insulin-resistant patients have been identified that possess a missense mutation in one of their insulin receptor alleles and the same nonsense mutation at codon 1000 in their second allele (Moncada et al., 1986; Kadowaki et al., 1990a; Kusari et al., 1991). The truncated receptors encoded by the allele containing the opal stop codon at position 1000 lack the consensus sequence for ATP binding as well as most of the tyrosine kinase domain. From studies carried out with lymphocytes and fibroblasts from these patients, it was unclear whether the truncated receptors were normally synthesized, processed, and transported to the cell surface. It was also unknown whether the truncated molecules had normal half-lives or if deletion of the C-terminal and tyrosine kinase domains resulted in altered receptor turnover. In addition, it was unknown whether, following insulin binding, the mutant receptors were phosphorylated by receptors synthesized from the other allele of the insulin receptor gene, thus allowing them to interact with cellular substrates involved in mediating insulin's actions. To address these points, we have characterized the A1000 truncated receptor by trans- fection of the mutant cDNA into NIH-3T3 cells. We report here structural and functional studies of recombinant human insulin receptors truncated at codon 1000, resulting in deletion of 343 carboxyl-terminal amino acids. This article not subject to U.S. Copyright. 'Published 1994 by the American Chemical Society