There is morphological and biochemical evidence that insulin is internalized in hepatocytes. The present study was designed to investigate the fate of the insulin receptor itself, subsequently to the initial binding step of the hormone to the hepatocyte plasma membrane . The insulin receptor was labeled with a ' 25 1-photoreactive insulin analogue (B2[2-nitro,4-azidophenylacetyl]des-Pheet -insulin) . This photoprobe was covalently coupled to the receptor by UV irradiation of hepatocytes after an initial binding step of 2-4 h at 15°C. At this temperature, only limited (-20%) internalization of the ligand occurred . In a second step, hepatocytes were resuspended in insulin-free buffer and further incubated for 2-4 h at 37°C. After 2 h at 37°C, no significant radioactivity could be detected in non-UV-irradiated cells, whereas 12-15% of the radioactivity initially bound remained associated to UV-irradiated cells. Morphological analysis after electron microscopy revealed that -70% of this radioactivity was internalized and preferentially associated with lysosomal structures . SDS PAGE analysis under reducing conditions revealed that most of the radioactivity was associated with a 130,000-dalton band, previously identified as the major subunit of the insulin receptor in a variety of tissues. Internalization of the labeled insulin-receptor complex at the end of the 37°C incubation was further demonstrated by its inaccessibility to trypsin . Conversely, at the end of the association step, the receptor (also characterized as a predominant 130,000-dalton species) was localized on the cell surface since it was cleaved by trypsin. We conclude that in hepatocytes the insulin receptor is internalized with insulin .Evidence has accumulated that insulin, following its binding to specific receptors located on the surface of target cells, is progressively internalized and degraded in intracellular structures (1-4) . Studies aimed at demonstrating insulin internalization mainly involved morphological techniques and the use of radiolabeled or fluorescently labeled insulin. Although the results have unequivocally shown that the hormone enters the cell, the fate of the receptor itself remains largely unknown. We therefore decided to examine this problem by taking advantage of a photoreactive insulin analogue to covalently label the insulin receptor in hepatocytes. The basic questions we wanted to address were: (a) is the receptor internalized with insulin and (b) is the receptor degraded during the internalization process? 82The biochemical and morphological data presented in this paper indicate that the insulin receptor, covalently bound to a photoreactive insulin analogue, is internalized and preferentially associated with lysosomal structures as molecular species similar to those present on the cell surface . MATERIALS AND METHODS Isolation and Incubation of HepatocytesHepatocytes were isolated from male Wistar rats (120-150 g) by collagenase dissociation of the liver as previously described (5). All experiments were carr...

We have followed the fate of cell surface insulin receptors in isolated rat hepatocytes by both a biochemical and a morphological approach. Hepatocytes were labeled with the photoreactive and biologically active l25I-labeled insulin analogue, [2-nitro-4-azidophenylacetylB2Ides-PheBi-insulin, under conditions that allow for minimal internalization (2 hr at 150C). Analysis of the cell-associated radioactivity by NaDodSO4/polyacrylamide gel electrophoresis under reducing conditions followed by autoradiography revealed the specific labeling of a major insulin receptor subunit with Mr 130,000 and a minor degradation product with Mr 125,000. When the cells were exposed at 150C to trypsin at the end of the association period, these two bands were no longer observed, indicating that the labeled receptors were at the cell surface. This trypsin sensitivity of the receptor disappeared within 30-60 min of incubation of the cells at 37C, reflecting the internalization ofthe hormone-receptor complexes. Over the subsequent 4 hr ofincubation, this was followed by a progressive reappearance ofthe receptor complexes at the cell surface, as indicated by the recovery of trypsin sensitivity of the labeled insulin receptors. An identical (both chronologically and quantitatively) journey of the insulin receptors was observed when the labeled material was studied by quantitative electron microscopic autoradiography. Thus, when the cells were incubated at 37C there was a rapid decrease (30-60 min) in the percentage ofautoradiographic grains associated with the plasma membrane, followed by a progressive increase in this percentage over the subsequent 4 hr ofincubation.In conclusion, using a biochemical and morphological approach to trace the photoaffinity-labeled insulin receptor, we have shown that the internalized hormone-receptor complex is recycled back to the cell surface.The occurrence of receptor recycling that follows receptor-mediated endocytosis has been described for various ligands, including low density lipoproteins (LDL) (1, 2), asialoglycoproteins (3), mannose glycoconjugates (4), lysosomal enzymes (5), and a2-macroglobulin (6,7). In all ofthese cases, the major role of the receptors is to mediate ligand internalization, either by supplying the cells with cholesterol (LDL) or by allowing the removal of injurious agents from extracellular fluids (lysosomal enzymes, mannose, or galactose terminal glycoproteins) (8). However, little information is available concerning the fate of membrane receptors that bind hormones or neurotransmitters and whose major function is to convey a signal to the target cell. Recent studies suggest that receptors for epidermal growth factor (9, 10) and for acetylcholine (11) are internalized but not recycled in the presence of the ligand.In the present study we have specifically labeled the insulin receptor in situ in isolated rat hepatocytes using a photoreactive and biologically active insulin analogue. (15). Cells were incubated in Krebs-Ringer bicarbonate (KR bicarbonate) buffer (pH 7...

The cellular fate of insulin receptors in isolated rat adipocytes was studied by using a biologically active photosensitive insulin derivative, 'B(2-nitro4-azidophenylacetyl)-~des-Phe B insulin (NAPA-DP-insulin), to photoaffinity label the insulin receptors. Insulin receptors specifically labeled with "SI-labeled NAPA-DP-insulin were identified by NaDodSO/polyacrylamide gel electrophoresis and autoradiography. Under nonreducing conditions, specific bands ofMr 330,000, 295,000, and 260,000 were identified; under disulfide reducing conditions, these were converted into Mr 125,000 and 90,000 subunits. When cells labeled at 16°C were immediately trypsinized, all of the receptor bands were degraded into lower molecular weight fragments, indicating that the labeled receptors were all on the cell surface. However, when the labeled cells were incubated at 370C for 1 hr prior to trypsin exposure, "30% of the receptors were found to be trypsin insensitive, indicating that this fraction was translocated intracellularly. Processing of the insulin receptors appeared to occur; incubation at 37C (but not at 16'C) resulted in generation of a Mr 115,000 component from the Mr 125,000 subunit as well as in the disappearance of the Mr 330,000 and 295,000 species. Inclusion of chloroquine during photoaffinity labeling at 16°C and during the subsequent incubation at 37C showed that this agent (i) increased the trypsin-insensitive (intracellular) receptor pool, (ii) blocked conversion of the Mr 125,000 subunit into the Mr 1,15,000 component, and (iii) prevented the disappearance of the Mr 330,000 and 295,000 species. These studies show that insulinreceptor complexes are internalized and processed intracellularly at a chloroquine-sensitive site(s).Several recent morphologic and biochemical studies have shown that, following initial cell surface binding, a portion of bound insulin is internalized by adsorptive endocytosis and subsequently degraded intracellularly (for review, see ref. 1). Also, it is suggested that the receptor is internalized along with insulin (2, 3) and that this process participates in the insulin-induced receptor loss (down regulation) observed in various cell types (4-7). However, since almost all of the reported studies have followed the fate of radiolabeled insulin, conclusions regarding the fate of the receptor itself have been based on indirect evidence. Thus, it becomes necessary to use experimental techniques by which the receptor can be followed directly. Recently, the techniques of chemical crosslinking (8, 9) and photoaffinity labeling (10-13) have been used to identify the insulin receptor and study its subunit structure in a variety of tissues. Of these techniques, receptor photoaffinity labeling is an attractive approach to studies ofthe dynamics ofinsulin-receptor interaction in living cells and of the metabolic fate of the receptor. This is because covalent attachment of insulin to its receptor occurs at the ligand binding site (without perturbation of other cellular proteins) and the timing...

The insulin receptor in isolated rat liver plasma membranes was covalently labeled with the photoreactive insulin analogue NB-29-[(4-azido-2-nitrophenyl)acetyl]insulin and solubilized with the nondenaturing detergent Triton X-100. The resulting protein-detergent complex was characterized by gel filtration on Sepharose 6B, sedimentation rate determination in linear sucrose gradients, and equilibrium isopycnic centrifugation in NaBr and CsCl. The labeled insulin receptor was found in two forms. The Strokes radii and s20,w's of the two receptor-detergent complexes (R1 and R2) were (mean +/- SEM) 7.08 +/- 0.04 and 3.62 +/- 0.05 nm and 10.45 +/- 0.04 and 6.54 +/- 0.15 S, respectively. The two forms appeared to have the same buoyant density, 1.285 +/- 0.002 g cm-3. The dissociation of R2 from R1, or its reaggregation, either with itself or with other unlabeled proteins, to give R1 proceeded without chemical modification. Mild reduction of disulfide bonds (1 mM 1,4-dithiothreitol) increased the dissociation of R2 from R1. These results indicate that the solubilized receptor binds significant amounts of detergents, that the insulin binding component of the receptor binds to other receptor components by hydrophobic interactions, and that one or more components of the insulin receptor contain intrachain disulfide bonds.

We have studied the functional and structural characteristics of insulin receptors on cultured rat hypothalamic cells. The receptors on these cells are specific for insulin, but have a lower binding affinity than that measured in nonneuronal tissues. Neither acute (2-h) nor long term (24-h) exposure of the hypothalamic cells to high insulin concentrations resulted in receptor down-regulation. However, insulin is internalized in these cells and accumulated in the presence of the lysomotropic agent chloroquine. Acute exposure to insulin does not alter initial rate of 2-deoxyglucose transport in hypothalamic cells, but does cause a stimulation of aminoisobutyric acid uptake. Photoaffinity labeling of the receptors of the hypothalamic cells with a biologically active photosensitive insulin revealed a major specifically labeled band of 115K mol wt and a minor band of 40K mol wt under disulfide-reducing conditions compared to bands of 125K and 90K mol wt seen after labeling of the insulin receptors of adipocytes. The receptor proteins in hypothalamic cells under nonreducing conditions (420K, 370K, and 310K mol wt) were also smaller than those in adipocytes. Thus, the insulin receptors of cultured hypothalamic cells differ from insulin receptors on peripheral target tissues in both functional and structural aspects.