Subcellular gradients of cytosolic free Ca2+ concentration, [Ca2+]i, are thought to be critical for the localization of functional responses within a cell. A potential but previously unexplored mechanism for the generation of gradients of [Ca2+]i is the accumulation of Ca2+ stores at the site of Ca2+ action. The distribution of the Ca2+ store markers Ca(2+)-dependent adenosine triphosphatase and calreticulin was investigated in resting and phagocytosing human neutrophils. Both proteins showed an evenly distributed fine granular pattern in nonphagocytosing cells, but became markedly concentrated in the filamentous actin-rich cytoplasmic area around the ingested particle during phagocytosis. This redistribution began at early stages of phagocytosis and did not depend on an increase in [Ca2+]i. Thus, accumulation of Ca2+ stores in a restricted area of the cell may contribute to the generation of localized increases in [Ca2+]i.

LOW density lipoprotein (LDL) and epidermal growth factor (EGF) bind to receptors on the surface of human fibroblasts and are internalized in coated vesicles. Each of the ligands has been studied separately by electron microscopy in human fibroblasts using ferritin-LDL as one visual probe and 1251-EGF as a second visual probe. A mutant strain of human fibroblasts (J.D.) has been described in which LDL does not localize to coated pits and hence is not internalized. Because LDL and EGF do not compete with each other for binding, in the current studies we coincubated the two ligands with normal and mutant cells to visualize their cellular fates. In normal fibroblasts ferritin-LDL and 12~I-EGF both bound preferentially to coated pits at 4°C and both ligands were internalized into endocytotic vesicles and lysosomes. Quantitative studies in normal cells showed that 75% of the coated pits and vesicles that contained 12SI-EGF also contained ferritin-LDL, indicating that both ligands enter the cell through the same endocytotic vesicles. In the LDL internalization-mutant J.D. cells, ferritin-LDL did not localize in coated pits and was not internalized, but ~2SI-EGF bound to coated pits and was internalized just as in normal fibroblasts.Low density lipoprotein (LDL) and epidermal growth factor (EGF) bind to specific receptors on the surface of human fibroblasts and are subsequently internalized by receptor-mediated endocytosis. When morphologic probes of the two ligands are studied separately in the form of ferritin-LDL (1-3) I~5I-LDL (4), or ~25I-EGF (5) at 4°C, both ligands localize preferentially to coated pits on the cell surface, and at 37°C both are internalized by the cells. By contrast, a mutant strain of fibroblasts (J.D.) binds LDL, but the ligand is not internalized because its receptors are not localized in coated pits (4, 6).Since a variety of different ligands appear to be internalized by receptor-mediated endocytosis, often by coated pits (7), it is of interest to know whether these receptors function through common or separate coated regions of membrane. By fluorescence microscopy, it has been reported that a2-macroglobulin and EGF are internalized by a common endocytotic vesicle pathway (8). Electron microscopy studies have shown that a2-macroglobulin and vesicular stomatitis-virus particles localize to the same coated pits and endocytotic vesicles (9). Similar observations were reported for az-macroglobulin and the lysosomal enzyme fl-galactosidase (10). Although these studies suggest that receptors for different ligands are not segregated into separate coated pits and vesicles, quantitative data on the extent of co-localization was not provided.In this study, we have taken advantage of the unique properties of the mutant J.D. fibroblasts together with the well established quantitative methodology offered by ferritin binding and 12~1 autoradiography to address three questions about the distribution of binding sites for LDL-ferritin and 125I-EGF in the same cell: (a) What is the distribution of EG...

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...

When '25I-labeled insulin ('25I-insulin) is incubated with 3T3-L1 adipocytes and cells processed for electron microscopic autoradiography, the ligand initially localizes preferentially to microvilli and coated pits. As a function of time and temperature, this initial preferential.localization to microvilli is lost, and the ligand is internalized by the cell. Serial sections of apparent coated vesicles near the cell surface indicate that about half of these structures are true vesicles and, therefore, intermediates in this receptor-mediated endocytotic process. With time, '251-insulin localizes to larger intracellular membranebounded structures. When cells are incubated with another ligand, cationic ferritin, that is taken up by adsorptive endocytosis, essentially the same structures are involved as for the endocytosis of '2I-insulin. The data suggest that specificity for receptor-mediated endocytosis is conferred by the. specific ligand receptor and possibly by ligand-induced receptor mobility in the plane of the plasma membrane. Other structures such as coated pits, coated vesicles, larger vesicles, and secondary lysosomes are common for different ligands.Direct morphologic studies have demonstrated that polypeptide hormones and their specific cell surface receptors are internalized by a variety of cell types (1-4). This receptor-mediated process provides a mechanism for removal of the ligand from the cell surface for inactivation and a mechanism by which cell surface receptors may be regulated.In the present investigation, we have tracked the morpho- with '25I-insulin (5 ng/ml) or cationic ferritin (0.75 mg/ml) for the times and temperatures specified. After the incubations, cells were washed in buffer at 4°C and fixed on the plate in 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 2 hr. The plates were then washed; and cells were postfixed in 0.1 M osmium tetroxide (pH 7.4), dehydrated in ethanol, and embedded in Epon. Thin sections were cut parallel to the plane of the culture and placed on copper grids.Grids containing '"I-insulin were then processed for autoradiography (6, 7).Sampling and Analysis of Data. The method ofsampling was similar to that as described (7,8). All The relationship ofautoradiographic grains to specific plasma membrane structures was assessed by the "hypothetical grain" method of Blackett and Parry (9, 10) and will be described in detail elsewhere (11) as applied to this type of study. In brief, the method analyzes the "real grains" (i.e., developed autoradiographic grains) in terms of their relationship to underlying structures versus the distribution of random or "hypothetical grains." If the real-grain distribution can be fitted to the hypothetical-grain distribution by a specially designed computer program, then the distribution is random. Failure ofreal grains to be fitted to the hypothetical-grain distribution represents preferential labeling ofa structure and is expressed by the term "grains per grid point. " The data also can be expressed in terms ofrelat...

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...