INSULIN-RECEPTORS INTERNALIZE BY A RAPID, SATURABLE PATHWAY REQUIRING RECEPTOR AUTOPHOSPHORYLATION AND AN INTACT JUXTAMEMBRANE REGION

The effect of receptor occupancy on insulin receptor endocytosis was examined in CHO cells expressing normal human insulin receptors (CHO/IR), autophosphorylation- and internalization-deficient receptors (CHO/IR(A1018)), and receptors which undergo autophosphorylation but lack a sequence required for internalization (CHO/IR(DELTA-960)). The rate of [I-125]insulin internalization in CHO/IR cells at 37-degrees-C was rapid at physiological concentrations, but decreased markedly in the presence of increasing unlabeled insulin (ED50 = 1-3 nM insulin, or 75,000 occupied receptors/cell). In contrast, [I-125]insulin internalization by CHO/IR(A1018) and CHO/IR(DELTA-960) cells was slow and was not inhibited by unlabeled insulin. At saturating insulin concentrations, the rate of internalization by wild-type and mutant receptors was similar. Moreover, depletion of intracellular potassium, which has been shown to disrupt coated pit formation, inhibited the rapid internalization of [I-125]insulin at physiological insulin concentrations by CHO/IR cells, but had little or no effect on [I-125]insulin uptake by CHO/IR(DELTA-960) and CHO/IR(A1018) cells or wild-type cells at high insulin concentrations. These data suggest that the insulin-stimulated entry of the insulin receptor into a rapid, coated pit-mediated internalization pathway is saturable and requires receptor autophosphorylation and an intact juxtamembrane region. Furthermore, CHO cells also contain a constitutive nonsaturable pathway which does not require receptor autophosphorylation or an intact juxtamembrane region; this second pathway is unaffected by depletion of intracellular potassium, and therefore may be independent of coated pits. Our data suggest that the ligand-stimulated internalization of the insulin receptor may require specific saturable interactions between the receptor and components of the endocytic system.