Changes in the patching and capping of insulin receptors under the influence of hormonal imprinting

Binding of fluorescein-isothiocyanate-(FITC)-labeled insulin was followed up in the function of time in Chang liver cells pretreated and not pretreated with insulin. The not pretreated cells showed patching, but no capping of the receptors during the period of study (60 min), whereas the insulin-pretreated cells showed indications of capping already after 10 min. Patching of the insulin receptors was particularly conspicuous at the sites of cell-cell contact (at the intercellular junctions). Supra-nuclear patching occurred earlier in the control cultures, and on it followed the fluorescence of the nuclear chromatin.     

Tyrosine phosphorylation/dephosphorylation controls capping of Fcgamma receptor II in U937 cells

In the capping of cell-surface receptors two stages can be distinguished: 1) clustering of the receptors (patching) induced by cross-linking with specific antibodies and 2) subsequent assembly of patches into a cap which is driven by the actin-based cytoskeleton. We found that patching of Fcgamma receptor II in U937 cells was correlated with tyrosine phosphorylation of certain proteins, most prominently those of 130, 110, 75 and 28 kDa. The phosphotyrosine-bearing proteins were accumulated at the receptor patches. Formation of the receptor caps was coincident with dephosphorylation of these proteins. Inhibition of protein tyrosine kinases with herbimycin A and genistein attenuated the protein tyrosine hyperphosphorylation and blocked capping in a dose-dependent manner. Phenylarsine oxide and pervanadate, inhibitors of protein tyrosine phosphatases, also suppressed capping of Fcgamma receptor II in a concentration-dependent fashion. Simultaneously, tyrosine hyperphosphorylation of proteins occurred. In the presence of the tyrosine kinase and phosphatase inhibitors the receptors were arrested at the patching stage. In contrast, okadaic acid, a serine/threonine phosphatase blocker, did not affect assembly of the receptor caps. The inhibitory effect of phenylarsine oxide was rapidly reversed by dithiols, 2,3-dimercapto-1-propanoldithiol and dithiotreitol, and was coincident with dephosphorylation of protein tyrosine residues. Extensive washing of pervanadate-exposed cells also resulted in progressive restoration of the cap assembly. Using streptolysin O-permeabilized cells we confirmed regulatory function played by dephosphorylation of tyrosine residues in capping of Fcgamma receptor II. Exogenous phosphatases, applied to permeabilized cells in which activity of endogenous tyrosine phosphatases was blocked, evoked dephosphorylation of protein tyrosine residues that was accompanied by recovery of capping ability in the cells.     

Polymerization of additional actin is not required for capping of surface antigens in B-lymphocytes

CH12 is a murine B-cell lymphoma whose surface immunoglobulin (sIg) and concanavalin A (Con A) receptors patch and cap readily. Actin may be involved in CH12 patching and capping, since fodrin and F-actin collect under the cap, and cytochalasin D inhibits sIg capping. We have examined the state of the actin cytoskeleton during patching and capping. A wide range of concentrations of rabbit anti-mouse antibody (RAM) and Con A were used to patch or cap CH12 cells. G-actin was quantitated by DNase I inhibition, F-actin was quantitated by fluorescence-activated cell sorter analysis of fluorescent phalloidin staining, and actin nucleation sites were measured by pyrene actin polymerization. None of these methods detected any significant changes in actin when compared to control cells or untreated cells, leading us to conclude that increased actin polymerization is not necessary for capping to occur. The significance of these data to the membrane flow and cytoskeletal models of capping is discussed.     

Binding of plasma membrane glycoproteins to the cytoskeleton during patching and capping is consistent with an entropy-enhancement model

Concentrations of concanavalin A that induced patching and capping of cell surface receptors on Dictyostelium discoideum also induce binding of the receptors to the cortical cytoskeleton, which was isolated by density-gradient centrifugation. The receptors were solubilized by deoxycholate, purified by affinity chromatography, and used to determine whether the receptors bound directly to the cytoskeletal protein, actin. As the concentration of actin was increased, many of the receptors became bound to purified filamentous rabbit muscle actin, even in the absence of concanavalin A. As in the ligation-induced binding of receptors to the cortical cytoskeleton in cells, concanavalin A induced much stronger binding of the purified receptors to filamentous actin. The results were consistent with a previously stated hypothesis that induction of receptor binding to the cytoskeleton during their patching and capping is driven by clustering the receptors, which reduces their translational entropy and by doing so enhances their avidity for the cytoskeleton.     

Lymphocyte activation and capping of hormone receptors

In this study both a ligand-dependent treatment [concanavalin A (Con A)] and a ligand-independent treatment [high-voltage pulsed galvanic stimulation (HVPGS)] have been used to initiate lymphocyte activation via a transmembrane signaling process. Our results show that both treatments cause the exposure of two different hormone [insulin and interleukin-2 (IL-2)] receptors within the first 5 min of stimulation. When either insulin or IL-2 is present in the culture medium, the stimulated lymphocytes undergo the following responses: (1) increased free intracellular Ca2+ activity; (2) aggregation of insulin or IL-2 receptors into patch/cap structures; (3) tyrosine-kinase-specific phosphorylation of a 32-kd membrane protein; and finally (4) induction of DNA synthesis. Further analysis indicates that hormone receptor capping is inhibited by (1) cytochalasin D, suggesting the involvement of microfilaments; (2) sodium azide, indicating a requirement for ATP production; and (3) W-5, W-7, and W-12 drugs, implying a need for Ca2+/calmodulin activity. Treatment with these metabolic or cytoskeletal inhibitors also prevents both the tyrosine-kinase-specific protein phosphorylation and DNA synthesis which normally follow hormone receptor capping. Double immunofluorescence staining shows that actomyosin, Ca2+/calmodulin, and myosin light-chain kinase are all closely associated with the insulin and IL-2 receptor cap structures. These findings strongly suggest that an actomyosin-mediated contractile system (regulated by Ca2+, calmodulin, and myosin light-chain kinase in an energy-dependent manner) is required not only for the collection of insulin and IL-2 receptors into patch and cap structures but also for the subsequent activation of tyrosine kinase and the initiation of DNA synthesis. We, therefore, propose that the exposure and subsequent patching/capping of at least one hormone receptor are required for the activation of mouse splenic T-lymphocytes.     

Capping of Con A receptors and actin distribution are influenced by disruption of microtubules in Dictyostelium discoideum

Capping of Concanavalin A (Con A) receptors can be inhibited in Dictyostelium by treatment of amoebae with the microtubular drug tubulozole. In cells that were incubated with Con A or with fluorescent Con A conjugate the capping process was completed in 30 min as could be demonstrated by fluorescence microscopy and Con A peroxidase labeling. In the presence of 10(-5) M tubulozole redistribution of the receptors did not proceed beyond a stage that can be characterized as patching. The effect of the drug on microtubule integrity was checked by electron microscopy and immunofluorescence of tubulin. Treatment resulted in shortening of the peripheral parts of the microtubules, in agreement with results described by other authors. Electron microscopy confirmed that the Con A receptor complexes remained on the plasma membrane and were not internalized. The distribution of F-actin in Con A-treated cells showed a pattern closely resembling that of Con A. Cells that were also treated with tubulozole remained spherical and did not resume significant directional movement until tubulozole was removed from the medium. It is concluded that microtubules are involved in the rearrangement of the microfilament network in moving cells.     

Evidence of the receptor nature of the binding sites induced in Tetrahymena by insulin treatment. A quantitative cytofluorimetric technique for the study of binding kinetics

Tetrahymena pyriformis GL cells pretreated (imprinted) and not pretreated with insulin showed dissimilar quantitative relations of FITC-insulin binding. Displacement of FITC-insulin by unlabelled insulin was considerably less in the control than in the imprinted series. The curve for saturation of the binding sites with FITC-insulin resembled a true saturation curve. The imprinted cells bound considerably more hormone in a shorter time than the control cells at identical levels of exposure. The dissociation of bound hormone from the imprinted cells increased over the control at 23 degrees C, and to a still greater degree at 4 degrees C. The effect of the pH of the medium on the dissociation of bound FITC-insulin also differed between the imprinted and not imprinted cells. Thus the proposed cytofluorimetric assay of binding kinetics demonstrated the actual conditions of receptor activity, and indicated that the induced insulin binding sites of Tetrahymena behaved similarly to 'classical' receptors.     

A T-lymphoma transmembrane glycoprotein (gp180) is linked to the cytoskeletal protein, fodrin

A major mouse T-lymphoma surface glycoprotein (gp180) has been identified by labeling cells with 125I and [3H]glucosamine. After ligand-induced receptor patching and/or capping, the amount of gp 180 in the membrane-associated cytoskeleton fraction increases in direct proportion to the percentage of patched/capped cells. There is a parallel increase in the amount of fodrin in the membrane-associated cytoskeleton fraction. Evidence is presented that gp180 is the same as or very similar to the T-lymphocyte-specific glycoprotein T-200. An immunobinding assay of Nonidet P-40-solubilized plasma membrane selectively co-isolates gp180 and fodrin. After induction of receptor rearrangement, double-label immunofluorescence reveals that fodrin accumulated directly beneath gp180 patches and caps. Membrane extraction with Triton X-114 followed by sucrose gradient centrifugation permits isolation of a gp180-fodrin complex with a 1:1 molar ratio and sedimentation coefficient(s) of approximately 20. This complex remains stable during isoelectric focusing and exhibits a pl in the range of 5.2-5.7. On the basis of our results we conclude that gp180, an integral membrane glycoprotein, and fodrin, a component of the membrane-associated cytoskeleton, are closely associated into a complex. Furthermore, we contend that, through fodrin's association with actin, this complex is of functional significance in ligand-induced patching and capping of gp180. We also propose that, through lateral interactions in the plane of the membrane, the gp180-fodrin complex might be responsible for linking other surface receptors to the intracellular microfilament network during lymphocyte patching and capping.     

Insulin receptor cycling and insulin action in the rat adipocyte

The possibility that the insulin receptor of adipocytes undergoes cycling was examined by a method involving pronase digestion at 12 degrees C, followed by insulin binding studies to determine receptor location and quantity. In the absence of insulin treatment, the amount of internal receptors (i.e. protected from pronase) was 10% of total receptor content. Following a 30-min insulin treatment (0.1 microM) at 37 degrees C, the internal receptor content increased 2-fold (206 +/- 12% of control, 100%). This effect was rapid, and maximum internalization was approached by 5 min of insulin treatment. Warming pronase-digested cells to 37 degrees C allowed the internal receptors to move to the cell surface. This movement was rapid also, and expansion of the internal pool by insulin pretreatment provided a 2.4-fold increase in the reinsertion of cell-surface receptors (238 +/- 28% of nontreated cells, 100%). Insulin-pretreated and nontreated cells had approximately 13 and 6%, respectively, of their original cell-surface receptor content, i.e. their content before pronase digestion. These receptors appeared intact after the cycling process, as judged by affinity labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the receptor and its binding subunit. The ability of the recycled receptor to respond to insulin was examined by studies of glucose incorporation into lipids and the inhibition of isoproterenol-stimulated lipolysis. Cells pretreated with insulin and allowed to recycle (e.g. 13% of normal receptor content) were 2-3-fold more responsive and 7-fold more sensitive to subsequent insulin stimulation than nontreated cells (e.g. 6% of normal receptor content), indicating that the recycled receptors are biologically active and coupled to cellular effector systems.     

A Possible role of the nucleus in cytochalasin B-Induced capping

The possible influence of the nucleus on Cytochalasin B (CB)-induced capping of antibodies to surface antigens on L cells SV40-3T3 and NRK La 334 cells was studied. The cap formation induced by CB, was generally localized opposite the nucleus which was displaced against the cell periphery. To be able to observe the nuclear membrane in relation to the capping process we have taken advantage of an antiserum specific for antigens in the nuclear membrane but lacking reactivity to the plasma membrane and intranuclear antigens. This approach indicated that the CB-induced capping caused an accumulation of nuclear membrane antigens in the area of the nucleus facing the cap. The CB-induced local accumulation of nuclear membrane antigens required intact cells and could not be induced by binding of antibodies to the nuclear membrane followed by exposure to CB. Whatever the basis for the CB-induced altered reactivity of the anti-nuclear membrane antibodies (folding of the nuclear periphery, for example) this result indicated that the nuclear membrane was affected by CB capping. The possible role of the nucleus in the CB-induced capping process was further investigated in enucleated cells. The results obtained indicate that such cells both when enucleated in suspension and adherent to a surface did not exhibit CB capping. This disappearance of CB capping did probably not reflect decreased cell viability, previous exposure of the cells to CB during the enucleation procedure or a decreased capacity of the enucleated cells to bind CB.     

Insulin and insulin-like growth factor I (IGF I) stimulate phosphorylation of a Mr 175,000 cytoskeleton-associated protein in intact FRTL5 cells

FRTL5 rat thyroid cells possess separate high affinity receptors for insulin and insulin-like growth factor I (IGF I) that undergo beta-subunit phosphorylation upon interaction with the specific ligand. Phosphorylation is rapid and dose-dependent and occurs primarily on tyrosine residues. Within 2 min, both insulin and IGF I also give rise to a Mr 175,000 phosphoprotein (pp175) that can be immunoprecipitated by anti-phosphotyrosine antibody (alpha-Tyr(P]. Phosphorylation of pp175 occurs on serine and threonine as well as tyrosine residues. When FRTL5 cells are solubilized with 1% Triton X-100, alpha-Tyr(P) immunoprecipitates phosphorylated insulin and IGF I receptors but little pp175 from the Triton-soluble fraction. After treatment of the Triton-insoluble portion with 1% sodium dodecyl sulfate at 100 degrees C, pp175 can be identified by immunoprecipitation with alpha-Tyr(P). The fraction of FRTL5 cells that remains after extraction of an attached monolayer with 1% Triton for 5 min at 22 degrees C contains most of the cytoskeleton and also nuclei. Extraction of this 32P-labeled cytoskeleton preparation with sodium dodecyl sulfate followed by alpha-Tyr(P) immunoprecipitation results in almost complete recovery of the pp175 content of the cells. When a nuclear fraction was prepared from FRTL5 cells by differential centrifugation, pp175 was not found in the nuclear pellet from labeled cells, but greater than 80% of pp175 was recovered in the supernatant. We conclude that pp175 is a common substrate for insulin and IGF I receptor kinases which, in FRTL5 cells, is associated with the cytoskeleton. It is suggested that phosphorylation of proteins associated with cytoskeletal elements could be involved in insulin and IGF I action in cells.     

Redistribution of polycations bound to lymphocytes

Summary The redistribution of surface-bound polycations on human lymphocytes and mouse-spleen lymphocytes was studied by fluorescence microscopy. A redistribution pattern after incubation with protamine, polylysine and DEAE-dextran resembling patching and capping processes was observed both at 20°C and at 4°C. The interaction between polycations and the cell surface is considered to represent non-specific binding to the membrane proteins. The redistribution process is regarded as precipitation processes. This is in accordance with the finding that no effect on the binding and redistribution of polycations was noticed in the presence of NaN₃, vinblastine sulphate, vincristine sulphate, colchichine, cytochalasin B or trypsin treatment. When incubation periods longer than 5 min. were employed beginning internalization of flourescent material is observed. Interaction with the nuclear membrane similarly resulted in rearrangements and capping.Sponsored by the Danish Cancer Society.     

Anti-idiotypic antibody identifies the cellular receptor of reovirus type 3

The binding and subsequent infectivity of reovirus to target cells are mediated by interaction with specific cell surface viral receptors. To gain a more detailed understanding of the biochemistry of the reovirus receptor and the cellular consequences of viral attachment, we have studied the binding of type 3 reovirus (Dearing strain) in a quantitative manner utilizing an antiidiotypic antibody probe. A syngeneic monoclonal antiidiotypic antibody (87.92.6) was prepared by immunization with hybridoma cells which secrete an antireovirus hemagglutinin-specific antibody. This antiidiotypic antibody was previously shown to specifically recognize the cell surface receptor for reovirus type 3. In this report, we demonstrate that antiidiotype mimicked reovirus tropism in binding to murine thymomas; antiidiotype inhibited the binding of reovirus to specific targets, but not the binding of anti-H-2; and cross linking of receptor-bound antiidiotype by antiimmunoglobulin induced patching, but not capping of reovirus receptors. Utilizing radiolabeled antiidiotype, we next quantitate the number of reovirus receptors on R1.1 and YAC thymoma cells and, finally, report on the preliminary identification of the reovirus receptor as a 67,000-Da membrane glycoprotein.     

Shunting of insulin from a retroendocytotic pathway to a degradative pathway by sodium vanadate

Adipocytes route internalized insulin through two major pathways, a degradative pathway and a retroendocytotic pathway. To examine whether sorting of incoming insulin-receptor complexes can be altered, we assessed the effect of vanadate on the intracellular processing of both insulin and insulin receptors. After cells were pretreated with vanadate (1 mM for 30 min at 37 degrees C), 125I-insulin was loaded into the cell interior. When the net efflux of insulin from cells into the medium was then monitored, vanadate was found to slow the efflux of insulin from a t1/2 of 6.2 min (controls) to 11 min. Since efflux reflects both the rapid extrusion of intact insulin and the slower release of degradative products, we proposed that vanadate diverts more insulin into the degradative pathway. Further evidence in support of this idea included the following: 1) when intracellular degradation of insulin was impaired by chloroquine, undegraded insulin accumulated faster within vanadate-treated cells, consistent with greater flux through a degradative pathway; 2) vanadate increased the percentage of degraded insulin released from cells from 61 and 72%; and 3) under steady-state binding conditions, more insulin resided in the cell interior of vanadate-treated cells (44.8% versus 34.5%), and the time required for the intracellular pool to reach equilibrium was prolonged (t1/2 of 5.5 min versus 4.0). Neither insulin internalization nor degradation was impaired by vanadate alone. In related studies Tris was found to inhibit insulin-mediated receptor recycling by only 10%, whereas in the presence of vanadate (plus Tris) almost all incoming insulin receptors were prevented from recycling. Vanadate alone had no effect on the ability of insulin receptors to recycle. Based on these results we conclude that: 1) vanadate shunts incoming insulin from a more rapid retroendocytotic pathway to a slower degradative pathway and diverts insulin receptors from a Tris-insensitive recycling pathway to one that can be completely inhibited by Tris; 2) these effects are selective, in that vanadate impairs neither insulin degradation nor receptor uptake and recycling. Considered together, these findings support the idea that a sorting mechanism exists for the intracellular routing of incoming insulin-receptor complexes.     

Insulin down-regulates insulin receptor number and up-regulates insulin receptor affinity in cells expressing a tyrosine kinase-defective insulin receptor

We examined the effect of insulin treatment on HTC cells transfected with large numbers of either normal insulin receptors (HTC-IR) or insulin receptors defective in tyrosine kinase (HTC-IR/M-1030). In both HTC-IR and HTC-IR/M-1030 cells, 20 h of insulin treatment (1 microM) at 37 degrees C resulted in a 65% decrease in the number of binding sites with a reciprocal 6-fold increase in affinity. In contrast, treatment with 10 nM insulin (20 h, 37 degrees C) also increased receptor affinity but had a smaller effect on the number of binding sites. 125I-Insulin binding to soluble receptors from HTC-IR and HTC-IR/M-1030 cells pretreated with insulin showed results similar to those obtained in intact cells. In both HTC-IR and HTC-IR/M-1030 cells, insulin enhanced insulin receptor degradation. In HTC-IR/M-1030 cells a 1-h incubation with insulin did not change receptor number and had only a small effect on receptor affinity; also there was no effect of insulin after a 20-h incubation at 15 degrees C. Inhibiting protein synthesis by pretreatment with cycloheximide (100 microM) did not block either the decrease in receptor number or the increase in receptor affinity. Both HTC-IR and HTC-IR/M-1030 cells exhibited a very slow rate of insulin and insulin receptor internalization and no differences were seen in this parameter when HTC-IR cells were compared to HTC-IR/M-1030 cells. These studies indicate, therefore, that in cells expressing kinase-defective insulin receptors, insulin down-regulates insulin receptor number via enhanced receptor degradation, and up-regulates receptor affinity. These effects were time- and temperature-dependent, but not dependent on new protein synthesis, and suggest that activation of tyrosine kinase may not be a prerequisite for certain mechanisms whereby insulin regulates its receptor.     

Immunocytochemical localization of receptor human chorionic gonadotropin complexes in rat leydig cells

Localization of receptor-bound human chorionic gonadotropin (hCG) in rat testis was studied by the peroxidase-antiperoxidase (PAP) complex method. The rats were injected with a single intravenous dose (1000 IU) of hCG. Three, 6, 12, and 24 hr after injection the testes were removed for localization of the hormone. The hormone localized to the periphery of the Leydig cells at all observation points. The intensity of the staining varied between the cells, suggesting that the number of receptors or the accessibility of the receptors to the circulating hormone varies from one cell to another. The staining surrounded the Leydig cells unevenly, but no progressive patching or capping was found. This observation suggests that hCG binds preferentially to the cell surface areas directed toward the capillaries. Compatible results were obtained with anti-hCG serum and with antisera against the hCG subunits. These results are consistent with previous observations that the luteinizing hormone (hCG) receptors accessible to the circulating hormone are located at the surface of the Leydig cells.     

Interactions between a lymphoma membrane-associated guanosine 5'-triphosphate-binding protein and the cytoskeleton during receptor patching and capping

In this study we have used several complementary techniques to isolate and characterize a lymphoma membrane-associated 41-kDa protein that shares a number of structural and functional similarities with the alpha i subunit of the guanosine 5'-triphosphate (GTP)-binding protein (e.g., Gi alpha-like protein). In addition, using permeabilized lymphoma cells, we have found that: 1) GTP or GTP-tau-S augments, and pertussis toxin inhibits, phospholipase C (PLC) activity and receptor capping; and 2) the addition of lymphoma 41-kDa Gi alpha-like protein stimulates PLC activity and receptor patching/capping, and reverses the inhibitory effect of pertussis toxin on both activity and receptor patching/capping. Additional cytochemical and biochemical data indicate that the lymphoma 41-kDa protein is closely associated with several cytoskeletal proteins (e.g., actin, myosin, and fodrin) all of which colocalize under receptor cap structures. Furthermore, both the 41-kDa-mediated phospholipase C activity and receptor patching/capping are inhibited by cytochalasin D (a microfilament disrupting drug) and W-7 drug (a calmodulin inhibitor). Together, these data provide strong evidence for a functional association between the lymphoma membrane cytoskeleton and the 41-kDa (Gi alpha-like) protein. Specifically, this association appears to be required for the activation of phospholipase C that results in inositol triphosphate production, subsequent internal Ca2+ release, and finally surface receptor patching and capping.     

Internalization of insulin receptors and HLA antigens in human hepatoma cells.

Human HepG2 hepatoma cells express a high number of insulin receptors. Growing cells exhibit 70% of their insulin receptors on the plasma membrane. Moreover, cell-surface insulin receptors form molecular complexes with class I major histocompatibility antigens, as determined by co-immunoprecipitation of the receptors by anti-class I monoclonal antibodies. On exposure to saturating concentrations of insulin, the hormone is rapidly internalized into a Pronase-resistant compartment. Internalization of insulin is accompanied by a rapid (t1/2 = 2-3 min) redistribution of insulin receptors from the cell surface to an intracellular compartment. On removal of insulin from the medium, functional receptors recycle back to the plasma membrane, where they can bind insulin again. With chronic exposure of HepG2 cells to insulin, the initial redistribution of receptors is followed by a slow (t1/2 = 9 h) down-regulation of the receptors. Finally, notwithstanding their interaction at the cell surface, insulin receptors and class I major histocompatibility antigens are internalized at different rates and with independent regulation.     

Ultrastructural analysis of the organization and distribution of insulin receptors on the surface of 3T3-L1 adipocytes: rapid microaggregation and migration of occupied receptors

Monomeric ferritin-insulin and high-resolution electron microscopic analysis were used to study the organization, distribution, and movement of insulin receptors on differentiated 3T3-L1 adipocytes. Analysis of the binding to prefixed cells showed that insulin initially occupied single and paired receptors preferentially located on microvilli. The majority of receptors (60%) were found as single molecules and 30% were pairs. In 1 min at 37% C, 50% of the receptors on nonfixed cells were found on the intervillous plasma membrane and more than 70% of the total receptors had microaggregated. By 30 min only 7% of the receptors were single or paired molecules on microvilli. The majority were on the intervillous membrane, with 95% of those receptors in groups. The receptor groups on the intervillous plasma membrane could be found in both noncoated invaginations and coated pits. The concentration of occupied receptors in the noncoated invaginations and the coated pits was similar; however, ten times more noncoated invaginations than coated pits contained occupied insulin receptors. The observations in this study contrast with those reported on rat adipocytes using identical techniques (Jarett and Smith, 1977). Insulin receptors on adipocytes were initially grouped and randomly distributed over the entire cell surface and did not microaggregate into larger groups. Insulin receptors on rat adipocytes were found in noncoated invaginations but were excluded from the coated pits. The differences in the organization and behavior of the insulin receptor between rat and 3T3-L1 adipocytes suggest that the mechanisms regulating the initial organization of insulin receptors and the aggregation of occupied receptors may be controlled by tissue-specific processes. Since both of these cell types are equally insulin sensitive, the differences in the initial organization and distribution of the insulin receptors on the cell surface may not be related to the sensitivity or biological responsiveness of these cells to insulin but may affect other processes such as receptor regulation and internalization. On the other hand, the microaggregates of occupied receptors on both cell types may relate to biological responsiveness.     

Lysis of virus-infected target cells by antibody-dependent cellular cytotoxicity. II. Reversibility of the heart inactivation of K cell killing and relationship of Fc receptor capping to lysis.

The heat inactivation of human blood mononuclear cells active in antibody-dependent cellular cytotoxicity (ADCC) is largely reversed after 24 hr in culture at 37 °C. The reactivation process is inhibited by actinomycin D, cycloheximide, and emetine but not by mitomycin-C, indicating that recovery requires RNA and protein synthesis but not DNA synthesis. The ability of lymphocytes to cap surface immunoglobulin (SIg) and IgG-Fc receptors (FcR) was also studied. As with ADCC effector cell activity, both SIg and FcR capping were abolished by heating, and the kinetics of inactivation was similar to that of the inactivation of ADCC effector activity. In addition, the heat inactivation of capping was reversible in culture and followed kinetics of reactivation similar to that of K cell reactivation. These results suggest the participation of heat-labile proteins at or near the surface of the effector cell, which are also apparently involved in the capping of surface receptors. Presumably these heat-labile proteins are membrane-associated enzymes, but they may also be cytoskeletal structures such as microfilaments or microtubules whose heat-sensitivity is currently unknown. The mounting of lethal hits may involve the same membrane machinery which is responsible for capping or a capping process itself.