We have characterized the internalization and down-regulation of the insulin receptor and nine receptors with mutations in the transmembrane (TM) domain and/or flanking charged amino acids to define the role of this domain in receptor cycling. When expressed in Chinese hamster ovary cells, all had normal tetrameric structure and normal insulin-stimulated autophosphorylation/kinase activity. Replacement of the TM domain with that of the platelet-derived growth factor receptor, insertion of 3 amino acids, and substitution of Asp for Val938 or of Ala for either Gly933 or Pro934 had no effect on internalization. Replacement of the TM domain with that of c-neu or conversion of the charged amino acids on the cytoplasmic flank to uncharged amino acids, on the other hand, resulted in a 40-60% decrease in insulin-dependent internalization rate constants. By contrast, substitution of Ala for both Gly933 and Pro934 increases lateral diffusion mobility and accelerates internalization rate. These changes in internalization were due to decreased or increased rates of redistribution of receptors from microvilli to the nonvillous cell surface. In all cases, receptor down-regulation and receptor-mediated insulin degradation paralleled the changes in internalization. Thus, the structure of the transmembrane domain of the insulin receptor and flanking amino acids are major determinants of receptor internalization, insulin degradation, and receptor down-regulation.

Recent studies have demonstrated that reduced insulin-stimulated muscle glycogen synthesis is the major cause of insulin resistance in patients with non-insulin-dependent diabetes mellitus (NIDDM). This reduced rate has been assigned to a defect in either glucose transport or hexokinase activity. However it is unknown whether this is a primary or acquired defect in the pathogenesis of NIDDM. To examine this question, we measured the rate of muscle glycogen synthesis and the muscle glucose 6-phosphate (G6P) concentration using 13C and 31P NMR spectroscopy as well as oxidative and nonoxidative glucose metabolism in six lean, normoglycemic offspring of parents with NIDDM and seven age/weight-matched control subjects under hyperglycemic (approximately 11 mM)-hyperinsulinemic (approximately 480 pM) clamp conditions. The offspring of parents with NIDDM had a 50% reduction in total glucose metabolism, primarily due to a decrease in the nonoxidative component. The rate of muscle glycogen synthesis was reduced by 70% (P 0.005) and muscle G6P concentration was reduced by 40% (P 0.003), which suggests impaired muscle glucose transport/hexokinase activity. These changes were similar to those previously observed in subjects with fully developed NIDDM. When the control subjects were studied at similar insulin levels (approximately 440 pM) but euglycemic plasma glucose concentration (approximately 5 mM), both the rate of glycogen synthesis and the G6P concentration were reduced to values similar to the offspring of parents with NIDDM. We conclude that insulin-resistant offspring of parents with NIDDM have reduced nonoxidative glucose metabolism and muscle glycogen synthesis secondary to a defect in muscle glucose transport/hexokinase activity prior to the onset of overt hyperglycemia. The presence of this defect in these subjects suggests that it may be the primary factor in the pathogenesis of NIDDM.

A 10-allele polymorphism was identified in rad (ras associated with diabetes), a gene that is overexpressed in non-insulin-dependent diabetes mellitus (NIDDM) muscle. The polymorphism, designated RAD1, consists of a variable number of trinucleotide repeats (GTT and ATT) located in the poly(A) region of an intronic Alu sequence. Based on the number of GTT and ATT repetitions, the alleles can be grouped into four classes (I-IV). RAD1 allele frequencies were determined in 210 NIDDM patients and 133 nondiabetic control subjects, all Caucasians. One allele (number 8, class III) accounted for > 80% of the chromosomes in both groups. However, an excess of minor alleles, all belonging to class I, II, or IV, was observed among NIDDM chromosomes (P 0.025), suggesting a possible association between RAD1 and NIDDM predisposition. To promote further studies to test the hypothesis that genetic variability at the rad locus contributes to NIDDM, we mapped rad on the human genome. Using the fluorescence in situ chromosomal hybridization technique, rad was unequivocally assigned to chromosomal band 16q22. In families that were informative for RAD1, the rad locus was mapped within a 3-cM region defined by the markers D16S265, D16S186, and D16S397 (logarithm of odds scores = 10.08, 10.9, and 10.84 at recombination fractions of 0.024, 0.001, and 0.03, respectively). The high degree of heterozygosity of these markers will allow large-scale family studies to be performed to test the presence of linkage between rad and NIDDM.

Activation of the insulin receptor, like other tyrosine kinase receptors, appears to require dimerization. We have shown previously that, even in the absence of insulin, full receptor activation can be induced by changes in the receptor transmembrane domain (TMD), suggesting that TMD dimerization is sufficient for receptor activation. To further understand the importance of the TMD in insulin receptor activation, we have inverted the entire TMD sequence including flanking basic amino acids, residue-for-residue. This mutation was predicted to alter the ability of a TMD alpha-helix to form homodimers and higher level aggregates. Despite apparently normal protein folding on either side of the membrane, this mutation caused ER retention and, for those receptors that reached the cell surface, blockade of insulin-stimulated kinase signal transmission. However, the signaling blockade could be overcome by proteolytic activation with trypsin. In contrast, shifting only the basic cytoplasmic residues to the opposite side of the TMD or mutation to neutral residues had no detectable effect on assembly, biosynthesis, topology, or signaling. These findings extend our previous observations to suggest that TMD interactions within the membrane are not only sufficient for receptor activation, but may be required. TMD interactions also appear to be necessary for oligomeric assembly and biosynthetic maturation of the insulin receptor.