J L Carpentier

125I-insulin binding to cultured human lymphocytes. Initial localization and fate of hormone determined by quantitative electron microscopic autoradiography.

Morphologic and biochemical studies indicate that the initial action of insulin is binding to a cell surface receptor. Whether further translocation of the hormone, or a product of the hormone, occurs is unclear and has not been investigated by direct means. To determine the fate of 125I-insulin bound to its receptor, we have examined the distribution of radioactivity by quantitative electron microscopic autoradiography. Cultured lymphocytes of the IM-9 cell line were incubated with 0.1 nM 125I-insulin at 15 degrees and 37 degreesC for incubation periods extending from 2 to 90 min. At 15 degreesC, grains localize to the plasma membane and there is no translocation as a function of time. At 37 degreesC, grains predominantly localize to the plasma membrane but there is a small shift in distribution to a distance of 300-700 nm from the plasma membrane. This small additional band component of irradiation extends to approximately to10--15% of the cell radius. When a morphometric analysis is applied to grains extending 300 nm and beyond from the plasma membrane, we find no preferential localization to any intracellular organelle. We interpret these data to indicate that in the cultured lymphocyte, labeled insulin initially localizes to the plasma membrane but as fuanction of time and increasing temperature there is a small but definite translocation of the hormone or a product of the hormone to a hihgly limited aea of the cell periphery.

Dual role of a dileucine motif in insulin receptor endocytosis.

Two leucines (Leu986 and Leu987) have recently been shown to take part in the control of human insulin receptor (HIR) internalization (Renfrew-Haft, C., Klausner, R. D., and Taylor, S. I. (1994) J. Biol. Chem. 269, 26286–26294). The aim of the present study was to further investigate the exact mechanism of this control process. Constitutive and insulin-induced HIR internalizations were studied biochemically and morphologically in NIH 3T3 cells overexpressing either a double alanine (amino acid residues 986–987) mutant HIR (HIR AA1) or HIR truncated at either amino acid residue 981 (HIR Δ981) or 1000 (HIR Δ1000). Data collected indicate that: (a) the three mutant HIR show a reduced association with microvilli as compared with HIR wild-type; (b) the two receptors containing the dileucine motif (HIR WT and HIR Δ1000) show the highest propensity to associate with clathrin-coated pits, independently of kinase activation; (c) the two receptors lacking the dileucine motif but containing two tyrosine-based motifs, previously described as participating in clathrin-coated pit segregation, associate with these surface domains with a lower affinity than the two others, (d) in the presence of the kinase domain, an unmasking of the tyrosine-based motifs mediated by kinase activation is required. These results indicate that the dileucine motif is not sufficient by itself, but participates in anchoring HIR on microvilli and that another sequence, located downstream from position 1000 is crucial for this event. This dileucine motif also plays a role in HIR segregation in clathrin-coated pits. This latter function is additive with that of the tyrosine-based motifs but the role of the dileucine motif predominates. Eventually, the clathrin-coated pit anchoring function of the dileucine motif is independent of receptor kinase activation in contrast to the tyrosine-based motifs.

Binding, internalization, and lysosomal association of 125I-glucagon in isolated rat hepatocytes. A quantitative electron microscope autoradiographic study.

When 125I-glucagon is incubated with freshly isolated rat hepatocytes and studied by quantitative electron microscope autoradiography, the labeled material localizes to the plasma membrane of the cell at early times of incubation of 20 degrees C; at later times of incubation at 20 degrees C, there is little further translocation of the labeled ligand. When incubations are carried out at 37 degrees C, the labeled material is progressively internalized by the cell after a brief delay. When the internalized radioactivity is further analyzed, it is found to associate preferentially with lysosome-like structures. When the cell-associated radioactivity is extracted, there is degradation of the ligand in incubations carried out at 37 degrees C. The events involved in the interaction of 125I-glucagon with the hepatocyte are similar to those previously described for labeled insulin in this cell. The process of binding, internalization, and lysosomal association appears to be a general process related to many polypeptide hormones and growth factors, and may represent the mechanism by which the specific binding of the ligand to the cell surface mediates the degradation of the ligand and the loss of its surface receptor.

Receptor mediated endocytosis of polypeptide hormones: Mechanism and significance

Abstract Polypeptide hormones bind to specific receptors on the surface of cells. Under certain conditions they localize to specific microdomains of the membrane, i.e., microvilli and coated pits. At physiologic temperature the ligand is internalized by a process of adsorptive endocytosis. This process involves several intracellular membrane bounded structures including coated vesicles, non-coated vesicles and lysosomal structures. These events provide a simple and general mechanism for removal of the ligand from the cell surface in order to terminate its signal. Linked to this process is a mechanism for surface receptor regulation. Thus, the concentration of hormone receptors on the cell surface is a function of the synthetic rate, internalization rate and the rate of recycling of surface membrane.

Redistribution of 125I-Insulin on the Surface of Rat Hepatocytes as a Function of Dissociation Time

In the present experiments, we have correlated the distribution of 125I-insulin on the surface of rat hepatocytes with the dissociation of 125I-insulin from the cell. When 125I-insulin interacts with isolated rat hepatocytes at 15°C, an increasing proportion of the bound ligand becomes nondissociable under the influence of acid pH (6.0), trypsin (0.5 mg/ml), or an excess of unlabeled insulin (10−6 M). Under these conditions, only a small percentage of the labeled material is internalized as determined by quantitative electron microscope (EM) autoradiography. This progressive nondissociability of the ligand parallels its movement from microvilli to coated pits and its progressive concentration in these later surface specializations. These data suggest that receptors in different domains of the plasma membrane may have different dissociation rates for the ligand.

The interaction of 125I-insulin with cultured 3T3-L1 adipocytes: quantitative analysis by the hypothetical grain method.

The murine 3T3-L1 fibroblast under appropriate incubation conditions differentiates into an adipocyte phenotype. This 3T3-L1 adipocyte exhibits many of the morphologic, biochemical, and insulin-responsive features of the normal rodent adipocyte. Using quantitative electron microscopic (EM) autoradiography we find that, when 125I-insulin is incubated with 3T3-L1 adipocytes, the ligand at early times of incubation localizes to the plasma membrane of the cell preferentially to microvilli and coated pits. When the incubation is continued at 37 degrees C, 125I-insulin is internalized by the cells and preferential binding to the villous surface is lost. With the internalization of the ligand, two intracellular structures become labeled, as determined by the method of hypothetical grain analysis. These include large clear, presumably endocytotic, vesicles and multivesicular bodies. Over the first hour of incubation the labeling of these structures increases in parallel, but in the second hour they diverge: the labeling of multivesicular bodies and other lysosomal forms continuing to increase and the labeling of large clear vesicles decreasing. At 3 hours limited but significant labeling occurs in small Golgi-related vesicles that have the typical distribution of GERL. The distinct morphologic features of this cell make it ideal for a quantitative morphologic analysis and allow for an unambiguous view of the sequence of events involved in receptor-mediated endocytosis of a polypeptide hormone. These events are likely to be representative of the processing of insulin by the mature rodent adipocyte.