Internalization and cycling of the T cell antigen receptor. Role of protein kinase C

The dynamics of the T cell antigen receptor on a murine antigen specific T cell hybridoma have been analyzed using a monoclonal anti-receptor antibody. When this antibody, A2B4-2, is bound to surface receptors, no internalization is seen at 4 degrees C. Upon warming to 37 degrees C, between 20 and 30% of the antibody molecules are internalized over 20-30 min as measured by sensitivity to external acid. This level of internalization is identical if monovalent Fab fragments are used. In contrast, cross-linking of the anti-receptor antibody with a second antibody leads to rapid internalization of 100% of prebound surface A2B4-2. Phorbol 12-myristate 13-acetate (PMA) leads to the rapid internalization of up to 65% of the surface A2B4-2 or A2B4-2 Fab fragments. This effect requires protein kinase C and can be completely inhibited by depleting this kinase from the cells by long term treatment with high doses of PMA. Pretreatment of the T cells with PMA leads to a 40-50% drop in surface T cell antigen receptor expression. Despite the loss of surface receptors, the uptake of A2B4-2 in PMA-treated cells at 37 degrees C is identical to that seen in control cells. The total uptake of A2B4-2 at 37 degrees C is 25-30% greater than the number of surface receptors in control cells and about 100-150% greater than the number of surface receptors in PMA-treated cells. At steady state the percentage of total A2B4-2 on the cell surface is 75% for control cells and 38% for PMA-treated cells. The good agreement of these numbers with the percent internalization of a cohort of surface receptors suggests that all receptors are constantly cycling. The effect of PMA is to alter the kinetic parameters of this cycling, thus changing the steady state distribution of receptors between the plasma membrane and internal, presumably endosomal compartments. Measurement of initial rates of internalization suggests that the PMA effect can be largely explained by an increase in the internalization rate constant.     

Ligand-regulated internalization and recycling of human beta 2-adrenergic receptors between the plasma membrane and endosomes containing transferrin receptors.

Agonist-regulated redistribution of human beta 2-adrenergic receptors was examined in 293 cells. A specific antiserum recognizing the carboxyl-terminal hydrophilic domain of the receptor was developed, characterized, and used for immunocytochemical localization of receptors in fixed cells by conventional fluorescence and confocal fluorescence microscopy. The beta-adrenergic agonist isoproterenol induced redistribution of receptors from the surface of cells into small (less than 1 micron diameter) punctuate accumulations which were detected in cells within 2 min of agonist addition. The time course of receptor redistribution paralleled that of receptor sequestration measured by ligand binding, and receptor redistribution was reversible in the presence of the beta-adrenergic antagonist alprenolol. Optical sections imaged through cells by confocal microscopy localized receptor accumulations within the cytoplasm. To address the question of receptor internalization further, a mutant receptor possessing an engineered antigenic epitope in the amino-terminal hydrophilic domain was constructed, transfected into cells, and localized using both a monoclonal antibody recognizing the epitope tag (receptor ectodomain) and an antiserum recognizing the carboxyl terminus (receptor endodomain). In untreated cells most receptor antigen was detected at the cell surface, as assessed by accessibility to ectodomain antibodies in unpermeabilized specimens. In isoproterenol-treated cells, however, little receptor antigen was detected at the cell surface. Punctate receptor accumulations present in isoproterenol-treated cells were labeled by antibodies only following permeabilization of cells, as expected if these receptor accumulations were intracellular. Finally, internalized beta-adrenergic receptors colocalized with transferrin receptors, which are markers of endosomal membranes. These data provide several lines of evidence establishing that beta-adrenergic receptors undergo ligand-regulated internalization, they suggest that internalized receptors may be recycled back to the cell surface, and they provide the first direct indication that these processes involve the same endosomal membrane system passaged by constitutively recycling receptors.     

Rapid and reversible disappearance of beta-adrenergic cell surface receptors

The water soluble beta-adrenergic ligand [3H]CGP-12177 was used to measure the cell surface receptors in intact cells. In two cell lines, C6 glioma and WEHI 7 lymphoma cells, -50% of the cell surface receptors disappear within minutes of incubation of the cells with isoproterenol. The receptors can still be detected in homogenates and reappear on the cell surface when cells are washed and reincubated at 37 degrees C. The data agree with a disappearance of the receptors from the cell surface by an agonist-mediated endocytosis.     

An ultrastructural study of thrombomodulin endocytosis: internalization occurs via clathrin-coated and non-coated pits.

The regulation of thrombomodulin (TM) expression has been reported to occur by several mechanisms. We have examined constitutive internalization of TM using immunofluorescent and electron microscopic (EM) methods. A cell model was developed to study this process by introducing TM DNA into COS-7 cells for expression. The recombinant TM was determined to behave similarly to native TM from human umbilical vein endothelial cells (HUVEC) with respect to M(r) and cell surface functional activity. The transfected cells expressed 8-100-fold more functional TM per cell than HUVEC. Immunofluorescent studies on these cells indicated that anti-TM antibody-TM complex was internalized in a time- and temperature-dependent manner, with internalization detectable within 10 minutes. When the cells were incubated at 4 degrees C with gold-labelled anti-TM antibody, most of the gold particles were surface bound and detected by EM as individual particles or clusters of 2 or 3 particles. Initiation of endocytosis for 10 to 60 minutes resulted in a redistribution of gold particles into small clusters predominantly in non-coated pits and rarely in clathrin-coated pits, subsequently in early endosomes, multivesicular bodies, and lysosomes. Similar studies were performed with gold-conjugated thrombin, demonstrating a similar route of intracellular processing. These studies provide ultrastructural evidence that the process of endocytosis of TM involves the participation of both clathrin-coated and non-coated pits and vesicles, but that the latter process predominates. Further structure/function studies are indicated using our cell model, since defects in the endocytic pathway of this important anticoagulant receptor may contribute to the development of thromboembolic disease.     

Conjugates of colloidal gold with native and acetylated low density lipoproteins for ultrastructural investigations on receptor-mediated endocytosis by cultured human monocyte-derived macrophages

The morphological aspects of the binding and internalization of low density lipoproteins (LDL) and acetylated low density lipoproteins (AcLDL) by cultured human monocyte-derived macrophages were investigated. For this purpose, LDL and AcLDL were conjugated to 20 nm colloidal gold particles. After incubation of the cells with the conjugated lipoproteins at 4 degrees C some LDL- or AcLDL-gold complexes were found to be attached to the cell surface, but without characteristic localization. However, after incubation of the cells at 8 degrees C with either LDL-gold or AcLDL-gold, lipoprotein-gold complexes were present in clusters on the plasma membrane, often in coated pits. Cells incubated at 37 degrees C for various time periods showed internalization of both LDL- and AcLDL-gold complexes via small coated and non-coated vesicles and processing of the complexes in smooth-walled endosomes. When the cells were pulse-chased with LDL- or AcLDL-gold for 30 min at 37 degrees C, the gold conjugates occurred in dense bodies, probably lysosomes. The results suggest that although native and modified LDL are reported to be metabolized differently by macrophages, the morphological aspects of the endocytosis of LDL and AcLDL by cultured human monocyte-derived macrophages are similar.     

Evidence for the appearance of an uncoupled form of the beta-adrenergic receptor distinct from the internalized receptor

Agonist treatment of C6-glioma cells induces two altered states in beta-adrenergic receptors, a low affinity for the hydrophilic antagonist CGP-12177 and a low affinity for agonists like isoproterenol. We present evidence that, in cells not treated to inhibit receptor internalization, the two properties occur with a different time course, the low affinity for isoproterenol preceding that for CGP-12177. In that the low affinity for CGP-12177 is due to the internalization of the receptor, the results indicate that uncoupling of the receptor, indicated by the low affinity for isoproterenol, occurs while the receptor is still located on the cell surface. Removal of the agonist leads to reappearance of the receptor to the plasma membrane followed by loss of the uncoupled state.     

Collection of insulin, EGF and alpha2-macroglobulin in the same patches on the surface of cultured fibroblasts and common internalization.

We have used video intensification microscopy to observe fluorescent derivatives of insulin, epidermal growth factor and alpha2-macroglobulin added to Swiss 3T3-4 cells. At 4 degrees C, each of these polypeptides binds diffusely to specific receptors on the cell surface. When the cells are warmed to 23 or 37 degrees C, the bound insulin epidermal growth factor or alpha2-macroglobulin rapidly forms patches on the cell surface and is internalized. Using fluorescein-labeled alpha2-macroglobulin and rhodamine-labeled derivatives of insulin and epidermal growth factor, we show that all three polypeptides are internalized within the same vesicles by a common pathway. The mechanism for the internalization of these molecules is discussed.     

Agonist-induced changes in beta-adrenergic receptors on intact cells

Competition by beta-adrenergic agonists and antagonists for 125I-pindolol binding sites on intact cells (1321N1 human astrocytoma and C62B rat glioma) was measured using short time binding assays as previously described (Toews, M. L., Harden, T. K., and Perkins, J. P. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 3553-3557). Preincubation of cells with agonists converted about half of the cellular beta-adrenergic receptors from a form exhibiting high affinity for the agonists isoproterenol and epinephrine and the antagonist sotalol to a form exhibiting much lower apparent affinity for these ligands in short time assays. Exposure to agonists did not alter the affinity of receptors for the antagonist metoprolol. This change in the ligand binding properties of the receptor was rapid (t1/2 = 1-2 min following a lag of about 0.5 min), reversible (t1/2 = 6-8 min), and dependent on the agonist concentration present during the preincubation (K0.5 = 15 nM for isoproterenol). Both isoproterenol and sotalol attained equilibrium with the high affinity receptors very rapidly but equilibrated only slowly with those receptors exhibiting low apparent affinity in short time assays. These results are interpreted in terms of a model which postulates that both the low apparent affinity in short time assays and the subsequent slow equilibration of hydrophilic ligands with these receptors result from agonist-induced internalization of a fraction of cell surface beta-adrenergic receptors. The relationship of this change in receptor binding properties to other aspects of agonist-induced desensitization of the beta-adrenergic receptor-coupled adenylate cyclase system is discussed.     

Activation, desensitization, and recycling of frog erythrocyte beta-adrenergic receptors. Differential perturbation by in situ trypsinization

We have utilized limited in situ trypsinization of the adenylate cyclase-coupled beta-adrenergic receptor of frog erythrocytes to probe the processes of receptor activation, desensitization, and recycling. Treatment of intact erythrocytes with trypsin (1 mg/ml) for 1 h at 20 degrees C converts all the receptor peptides (identified by photoaffinity labeling with p-azido-125I-benzylcarazolol) from a Mr approximately 58,000 to a Mr approximately 40,000 species. Nonetheless, the trypsinized beta-adrenergic receptors bind agonists and antagonists with unaltered affinity and with no change in the number of binding sites. Moreover, the ability of the proteolyzed receptors to interact with the nucleotide regulatory protein to form a high affinity guanine nucleotide-sensitive state and to activate adenylate cyclase were also unaltered. However, upon exposure of intact cells to the agonist isoproterenol, trypsinized beta-adrenergic receptors were more rapidly and more completely cleared from the plasma membranes ("down-regulated") than untrypsinized receptors. Whereas down-regulated receptors from nontrypsinized cells appear to recycle to the cell surface after removal of the agonist, internalized trypsinized beta-adrenergic receptors do not recycle to the plasma membrane and appear to be degraded within the cell. Moreover, when internalized receptors, recovered in a light vesicle fraction, were fused with a heterologous adenylate cyclase system, untreated but not trypsinized receptors reconstituted catecholamine stimulation of the enzyme. These data suggest that the beta-adrenergic receptor contains a trypsin-sensitive site which is exposed on the outer surface of the plasma membrane. Proteolysis at this site releases a fragment which though not critically involved in either ligand binding or "effector coupling" might be important for anchoring the receptors in the plasma membrane. These data also suggest that in situ proteolysis of the receptors might serve as a physiological trigger for their internalization and degradation.     

Direct demonstration of rapid insulin-like growth factor II Receptor internalization and recycling in rat adipocytes. Insulin stimulates 125I-insulin-like growth factor II degradation by modulating the IGF-II receptor recycling process

The photoactive insulin-like growth factor (IGF)-II analogue 4-azidobenzoyl-125I-IGF-II was synthesized and used to label specifically and covalently the Mr = 250,000 Type II IGF receptor. When rat adipocytes are irradiated after a 10-min incubation with 4-azidobenzoyl-125I-IGF-II at 10 degrees C and immediately homogenized, most of the labeled IGF-II receptors are associated with the plasma membrane fraction, indicating that receptors accessible to the labeling reagent at low temperature are on the cell surface. However, when the photolabeled cells are incubated at 37 degrees C for various times before homogenization, labeled IGF-II receptors are rapidly internalized with a half-time of 3.5 min as evidenced by a loss from the plasma membrane fraction and a concomitant appearance in the low density microsome fraction. The low density microsomes were previously shown to contain intracellular membranes (Oka, Y., and Czech, M.P. (1984) J. Biol. Chem. 259, 8125-8133). The steady state level of cell surface IGF-II receptors in the presence or absence of IGF-II, measured by the binding of anti-IGF-II receptor antibody to cells, remains constant under these conditions, demonstrating that IGF-II receptors rapidly recycle back to the cell surface at the same rate as receptor internalization. Using the above methodology, it is shown that acute insulin action: 1) increases the steady state number of cell surface IGF-II receptors; 2) increases the number of ligand-bound IGF-II receptors that are internalized per unit of time, as evidenced by a large increase in the photolabeling of intracellular membrane IGF-II receptors when cells are incubated at 37 degrees C with insulin and 4-azidobenzoyl-125I-IGF-II prior to photoactivation; and 3) increases the rate of cellular 125I-IGF-II degradation by a process that is blocked by anti-IGF-II receptor antibody. The results indicate that the action of insulin to elevate the steady state number of cell surface IGF-II receptors leads to an increased internalization flux of IGF-II-bound receptors, mediating increased IGF-II uptake and degradation.     

Interaction of high density lipoprotein with its receptor on cultured fibroblasts and macrophages. Evidence for reversible binding at the cell surface without internalization

Cultured extrahepatic cells possess a specific high affinity receptor for high density lipoprotein (HDL) that is induced by cholesterol delivery to cells. Current results suggest that HDL receptors on cultured human fibroblasts and mouse peritoneal macrophages promote reversible binding of HDL to the cell surface without internalization of lipoprotein particles. When 125I-HDL3 was bound to cultured cells at 0 degrees C and then warmed to 37 degrees C after removal of unbound lipoprotein, most of the cell surface-bound HDL was released rapidly (t1/2 = 3 min) into the medium without entering a cellular pool that was inaccessible to digestion by trypsin at 0 degrees C. This lack of internalization of HDL was evident under conditions where internalization of 125I-low density lipoprotein and 125I-transferrin were readily detected. When cells were exposed to 125I-HDL3 at 37 degrees C, only a trace amount of iodinated apoprotein remained associated with cells after treatment of cells with trypsin. Fibroblasts treated with medium containing increasing concentrations of cholesterol exhibited a dose-dependent increase in reversible, trypsin-sensitive binding of 125I-HDL3 at 37 degrees C without an attendant increase in trypsin-resistant binding. These results suggest that reversible binding of HDL to its cell-surface receptor without subsequent endocytosis of receptor-HDL complexes is the mechanism by which HDL receptors facilitate cholesterol transport from cells.     

Evidence for the rapid internalization and recycling of lutropin receptors in rat testis Leydig cells.

A study into the binding of 125I-human chorionic gonadotropin (hCG) to the lutropin (LH) receptor in rat testis Leydig cells, and subsequent internalization of the hormone-receptor complex, has been carried out. The results show that there is rapid internalization of the hormone-receptor complex; 240 receptors/cell (from a total of approx. 4000 receptors/cell) were internalized each minute in the first hour after exposure to hCG. Radioactivity was released from the cell 1 h after internalization and was found to be associated with highly degraded hCG. The endocytic process was found to have two temperature-sensitive steps. At 4 degrees C, movement of the hormone-receptor complex inside the cell did not occur, and at 21 degrees C hormone accumulated within the cytoplasm but was not degraded or released from the cell. At 34 degrees C, internalization, degradation and loss of the degraded hormone from the cell occurred. These processes appeared to reach a steady state after 2 h. Even though there is rapid internalization of the hormone-receptor complex following exposure to hCG, the binding sites on the cell surface were maintained for at least 4 h. The number of binding sites on the cell surface was not decreased by a protein synthesis inhibitor but was reduced to undetectable levels by monensin. This compound inhibits acidification of endocytic vesicles, which is known to be an important prerequisite to receptor cycling. It is concluded that, in the rat testis Leydig cells, following binding of hCG to the LH receptor there is rapid internalization of the complex and that recycling of the receptor occurs to the cell surface. This process may be essential in maintaining the capacity of the Leydig cell to bind fresh hormone.     

Metabolism of photoaffinity-labeled insulin receptors by adipocytes. Role of internalization, degradation, and recycling

Insulin receptors on isolated rat adipocytes were photoaffinity-labeled with a biologically active photo-derivative of insulin (iodinated B2 (2-nitro-4-azidophenylacetyl)-des- PheB1 -insulin) in order to study the metabolism of surface receptors after binding insulin. Adipocytes were incubated with iodinated B2 (2-nitro-4-azidophenylacetyl)-des- PheB1 -insulin (40 ng/ml) at 16 degrees C until specific binding reached equilibrium, subjected to photolysis, and then incubated at 37 degrees C to follow the metabolism of the covalent insulin-receptor complexes. Susceptibility of labeled insulin receptors to tryptic digestion was used to distinguish between receptors on the cell surface and those inside the cell. Following incubation of photoaffinity-labeled adipocytes at 37 degrees C, there was an initial rapid loss of insulin receptors from the cell surface. The internalization of insulin receptors occurred at a significantly faster rate than the loss of receptors from the cell, resulting in an accumulation of intracellular receptors. The proportion of surface-derived receptors inside the cell reached an apparent steady state after 30 min and represented about 20% of the labeled receptors originally on the cell surface. Chloroquine had no effect on the internalization of insulin receptors but inhibited their degradation. Cycloheximide inhibited both internalization and degradation of insulin receptors. After 60 min at 37 degrees C, the disappearance of insulin receptors from the cell surface slowed markedly and the overall loss of insulin receptors from the cell was minimal. If chloroquine was added at this time, there was a marked increase in the loss of receptors from the cell surface with a concomitant 2-fold increase in the intracellular pool of surface-derived receptors. From these observations, we conclude that 1) internalization is not rate-limiting in insulin receptor degradation, 2) chloroquine has no effect on the internalization of insulin receptors but inhibits the intracellular degradation of receptors, 3) cycloheximide interferes with both the internalization and degradation of insulin receptors, and 4) the plateau in the loss of labeled receptors from the cell surface after 60 min at 37 degrees C could be due to a new steady state balance between internalization and recycling of photoaffinity-labeled receptors.     

Redistribution of cell surface transferrin receptors prior to their concentration in coated pits as revealed by immunoferritin labels

Summary Immunocytochemistry has been used to study distribution of cell surface transferrin receptors in erythroid, leukemic (K562) cells. The cells were fixed and labelled with monoclonal (OKT-9) anti-transferrin receptor antibodies; the antibody-labelled receptors were then detected by either immunofluoresceinor immunoferritin-antimouse-antibody conjugates. Typically, the immunoferritin labels were distributed diffusely at the non-coated regions of the cell surface as well as concentrated in the clathrincoated pits. To examine further this pattern of distribution, cells were labelled at 0° C and then warmed to 37° C for zero to 30 min prior to fixation. The majority of the immunoferritin labels were initially dispersed in small groups at the non-coated regions of the cell surface (mean = 6 immunoferritin labels/cluster), but larger groups were common subsequent to incubation at 37° C (mean = 13 immunoferritin labels/cluster). However, the size of immunoferritin labels in the coated pits was unchanged (mean = 12 immunoferritin labels/pit). Immunoferritin labels were typical in coated and uncoated vesicles l min after warming to 37° C, but common in endosomes, multivesicular bodies and lysosomes by 30 min. It appears that single cell-surface receptors form large aggregates prior to their concentration in coated pits. Coated vesicles, uncoated vesicles, and endosomal vacuoles may together form the non-lysosomal compartment where the internalized receptors might be dissociated from the ligands (antibodies).     

Role of the coated endocytic vesicle in the uptake of receptor-bound low density lipoprotein in human fibroblasts.

125I-labeled and ferritin-labeled low density lipoprotein (LDL) were used as visual probes to study the surface distribution of LDL receptors and to examine the mechanism of the endocytosis of this lipoprotein in cultured human fibrobasts. Light microscopic autoradiograms of whole cells incubated with 125I-LDL at 4 degrees C showed that LDL receptors were widely but unevenly distributed over the cell surface. With the electron microscope, we determined that 60-70% of the ferritin-labeled LDL that bound to cells at 4 degrees C was localized over short coated segments of the plasma membrane that accounted for no more than 2% of the total surface area. To study the internalization process, cells were first allowed to bind ferritin-labeled LDL at 4 degrees C and were then warmed to 37 degrees C. Within 10 min, nearly all the surface-bound LDL-ferritin was incorporated into coated endocytic vesicles that were formed by the invagination and pinching-off of the coated membrane regions that contained the receptor-bound LDL. With increasing time at 37 degrees C, these coated vesicles were observed sequentially to migrate through the cytoplasm (1 min), to lose their cytoplasmic coat (2 min), and to fuse with either primary or secondary lysosomes (6 min). The current data indicate that the coated regions of plasma membrane are specialized structures of rapid turnover that function to carry receptor-bound LDL, and perhaps other receptor-bound molecules, into the cell.     

Hemopexin joins transferrin as representative members of a distinct class of receptor-mediated endocytic transport systems

Receptor-mediated transport of heme by hemopexin in vivo and in vitro results in catabolism of heme but not the protein, suggesting that intact apohemopexin recycles from cells. However, until now, the intracellular transport of hemopexin by receptor-mediated endocytosis remained to be established. Biochemical studies on cultured human HepG2 and mouse Hepa hepatoma cells demonstrate that hemopexin is transported to an intracellular location and, after endocytosis, is subsequently returned intact to the medium. During incubation at 37 degrees C, hemopexin accumulated intracellularly for ca. 15 min before reaching a plateau while surface binding was saturated by 5 min. No internalization of ligand took place during incubation at 4 degrees C. These and other data suggest that hemopexin receptors recycle, and furthermore, incubation with monensin significantly inhibits the amount of cell associated of heme-[125I]hemopexin during short-term incubation at 37 degrees C, consistent with a block in receptor recycling. Ammonium chloride and methylamine were less inhibitory. Electron microscopic autoradiography of heme-[125I]hemopexin showed the presence of hemopexin in vesicles of the classical pathway of endocytosis in human HepG2 hepatoma cells, confirming the internalization of hemopexin. Colloidal gold-conjugated hemopexin and electron microscopy showed that hemopexin bound to receptors at 4 degrees C is distributed initially over the entire cell surface, including microvilli and coated pits. After incubation at 37 degrees C, hemopexin-gold is located intracellularly in coated vesicles and then in small endosomes and multivesicular bodies. Colocalization of hemopexin and transferrin intracellularly was shown in two ways. Radioiodinated hemopexin was observed in the same subcellular compartment as horseradish peroxidase conjugates of transferrin using the diaminobenzidine-induced density shift assay. In addition, colloidal gold derivatives of heme-hemopexin and diferric transferrin were found together in coated pits, coated vesicles, endosomes and multivesicular bodies. Therefore, hemopexin and transferrin act by a similar receptor-mediated mechanism in which the transport protein recycles after endocytosis from the cell to undergo further rounds of intracellular transport.     

Use of cell fusion techniques to probe the mechanism of catecholamine-induced desensitization of adenylate cyclase in frog erythrocytes

The catecholamine-sensitive adenylate cyclase system appears to be comprised of at least three components; the beta-adrenergic receptor (R component), the catalytic unit of adenylate cyclase (C component) and a nucleotide regulatory protein (N component), responsible for mediating the effects of guanine nucleotides on the system. Cell fusion techniques were used to investigate the role of these three components in the process of homologous desensitization in the frog erythrocyte. Dicyclohexylcarbodiimide (DCCD) was used to inhibit beta-receptor function in one population of frog erythrocytes, whilst phenyl glyoxal was employed to inactivate the N and C components in a second population of frog erythrocytes. Using Sendai virus to fuse the two types of modified cell, heterologous beta-adrenergic receptor-adenylate cyclase systems were constructed which contained components from each cell type. When beta receptors from cells previously desensitized to catecholamines were coupled to N-C components derived from fresh erythrocytes, the resulting hybrid exhibited a densitized response to isoproterenol. By contrast, when beta-adrenergic receptors from fresh cells were coupled to N-C components derived from desensitized erythrocytes, no decreased responsiveness to isoproterenol was apparent in the hybrid. That this resensitization was the result of the addition of fresh beta-adrenergic receptors was demonstrated in a control experiment. Frog erythrocytes were desensitized simultaneously to catecholamines and prostaglandin E1 and modified with DCCD which inactivates the beta-adrenergic receptor but not the prostaglandin receptor. When fresh beta-adrenergic receptors were supplied by cell fusion to these doubly desensitized erythrocytes, only the beta-adrenergic response was restored to control levels. The response to prostaglandin remained desensitized in the hybrids, indicating that the observed resensitization of catecholamine-stimulated adenylate cyclase activity was specific and was due to the addition of fresh beta-adrenergic receptors. These data suggest that in the frog erythrocyte, homologous desensitization is primarily the result of receptor-related alterations.     

Insulin increases the cell surface concentration of alpha 2-macroglobulin receptors in 3T3-L1 adipocytes. Altered transit of the receptor among intracellular endocytic compartments

The present study shows that insulin causes an increase in the binding of alpha 2-macroglobulin (alpha 2M) to 3T3-L1 adipocytes. Scatchard analysis of the binding at 4 degrees C indicated an approximate 2-fold increase in the number of alpha 2M binding sites, with no change in the apparent affinity of the receptor. In addition, a 2-3-fold increase in the binding of monoclonal antibody 2C6, which recognizes a component of the alpha 2M receptor, was found in cells treated at 37 degrees C with insulin and then KCN to inhibit receptor endocytosis. An increased cellular accumulation of alpha 2M was also observed in response to insulin. Interestingly, the increase in the rate of accumulation of alpha 2M was significantly smaller than the increase in the number of alpha 2M receptors on the cell surface, suggesting that the rate of ligand internalization or subsequent processing is altered in response to insulin. Ultrastructural analysis of the internalization pathway of the alpha 2M receptor was performed using colloidal gold-coupled 2C6 monoclonal antibody. Control cells incubated for 20 min at 37 degrees C with the gold-conjugated antibody displayed 40% of cellular gold particles on the cell surface and 60% within intracellular structures. In insulin-treated cells this proportion was reversed, with 64% of the particles being found on the cell surface, and only 36% within intracellular structures. Significant differences in the distribution of gold particles among intracellular structures were detected between control and insulin-treated cells. Whereas in control cells, 18% of the total cellular gold particles internalized into tubulovesicles and multivesicular bodies, in insulin-treated cells only 3% of the gold particles were found within these structures. These data indicate that the movement of this receptor between endocytic compartments is altered in response to insulin, and suggest that the effect of insulin to increase the cell surface concentration of alpha 2M receptors and the accumulation of alpha 2M is due, at least in part, to alterations in the endocytic portion of the receptor recycling pathway.     

Concanavalin A can trap insulin and increase insulin internalization into cells cultured in monolayer

When rat hepatoma cells (R-Y121B) were incubated with insulin at 37 degrees C, concanavalin A increased insulin internalization into cells. When R-Y121B cells were first incubated with labeled insulin at 4 degrees C then with concanavalin A at various concentrations at 37 degrees C, the total cellular radioactivity was much higher at high lectin concentrations than at low lectin concentrations. This increase was not only due to an increase in insulin internalization into cells but also to an increase in insulin binding to cell surfaces. Concanavalin A can trap insulin on the insulin receptors - a "trapping" effect. It has been concluded that insulin and concanavalin A binding sites are very close to each other on the insulin receptors.     

Internalization of the fibronectin receptor is a constitutive process

Using a monoclonal antibody specific for the hamster fibronectin receptor (FnR), we have demonstrated that a portion of the CHO cell FnR population is constitutively endocytosed. Three independent techniques were used to study the internalization: 1) after saturation binding of an anti-FnR antibody (PB1) to cells at 4 degrees C, internalization was initiated by warming to 37 degrees C, and then acid/salt elution of membrane-bound ligand was used to quantitate the internalized 125I-PB1; 2) cell vesicular traffic was pharmacologically disrupted with monensin or chloroquine, and the subsequent reduction of the cell surface pool of FnR was monitored; and 3) selective immunoprecipitation was used to separate surface and internalized 125I-labeled FnR. These experiments indicate that about 30% of the cell surface FnR is endocytosed with a t1/2 of 7 min and that this internalization occurs regardless of the ligation state of the receptor. Other observations indicate that the larger fraction of the cell surface FnR pool (70-75%) is apparently shed from the cell upon ligation with antibody at 37 degrees C. This process occurs much more slowly than receptor internalization and leads to an overall reduction in the amount of cell surface FnR. Our results suggest physically or chemically distinct populations of FnR, one of which is unavailable for internalization and recycling.