Interferon-alpha down-regulates insulin receptors in lymphoblastoid (Daudi) cells. Relationship to inhibition of cell proliferation

The Daudi line of human lymphoblastoid cells requires insulin and transferrin for growth in serum-free medium and is highly sensitive to the inhibitory effect of human leukocyte interferon (IFN-alpha) on cell proliferation. A variant subline of Daudi cells, which is resistant to the antiproliferative action of IFN-alpha, also has been grown in serum-free medium containing insulin and transferrin. The proliferation of IFN-sensitive and -resistant Daudi cells is dependent on the occupancy of insulin receptors, with optimal cell proliferation observed at high receptor occupancy (nearly 100%). No evidence was found for receptors for insulin-like growth factor I on Daudi cells. IFN treatment of IFN-sensitive cells decreased the capacity of the cells to bind 125I-insulin. The altered binding capacity was due to diminished specific, lower affinity insulin binding, as detected at high 125I-insulin concentrations. Higher affinity insulin binding was not altered by IFN. Insulin binding was also reduced in detergent-solubilized extracts from IFN-treated sensitive Daudi cells and the magnitude of the effect was comparable to that observed in intact cells. This indicates that the total number of insulin binding sites (surface + internal) is decreased in IFN-treated sensitive cells. Insulin binding to IFN-sensitive cells decreased linearly with time between 6 and 48 h from the addition of IFN. The effect on lower affinity insulin binding developed more rapidly than the inhibitory effect of IFN on cell proliferation. The insulin-binding capacity of Daudi cells resistant to the antiproliferative effect of IFN was unaffected by IFN, despite the fact that these cells contain as many cell surface IFN receptors as sensitive cells. These observations raise the possibility that lower affinity insulin binding is important in the growth-promoting actions of insulin.     

Ecdysteroid receptors in a tumorous blood cell line of Drosophila melanogaster

The tumorous Drosophila melanogaster blood cell line BII has been studied for evidence for the presence of ecdysteroid receptors. The [³H]ponasterone A (pon A)* used in this study has been extensively purified, and the location of the tritium in the molecule has been partially determined. BII cells do not metabolise ecdysteroids. Intact cells demonstrate a considerable specific uptake of [³H]pon A which is saturable, apparently showing two specific components: a very high affinity component (K_D = 0.3 nM) and a high affinity component (K_D = 2 nM). The specific binding of [³H]pon A to whole cells is compatible with unlabelled ecdysteroids, but not with mammalian steroid hormones. The association rate constant (k_a) for [³H]pon A was determined to be 3 × 10⁷M^?1min^?1 at 21 °C, while the dissociation rate constant (k_d) for the specifically bound [³H]pon A was found to be 4.4 × 10^?3/min. Together, the kinetic rate constants yield a value of 0.15 nM for the K_D. The receptors have been partially characterised in a cell-free extract prepared by sonification of the cells. The optimum pH for extraction and hormone binding is 8.2. Scatchard plots of binding data indicate that the cell-free extract also contains two high affinity specific binding components (k_D = 0.1 nM and K_D = 1 nM). The hgih affinity binders are macromolecular, as shown by chromatography on Sephadex G-25, and are susceptible to protease digestion, heat, and treatment with N-ethylmaleimide. Sucrose density centrifugation of the labelled receptor shows one peak at approximately 6S. The stability of the receptor preparation has been studied and conditions have been empirically determined (10% w/v sucrose, 25 mM dithioerthreitol, and 10 mM citrate), whereby the binding capacity of the unlabelled receptor is stable for at least 8 weeks if frozen at ?20°C.     

Histamine H-1 receptors on rat peritoneal mast cells

In order to characterize the receptor subtype involved in histamine stimulation of increased cyclic AMP levels in rat mast cells with consequent impairment of anaphylactically induced mediator release, the binding of the H-1 receptor antagonist [³H] pyrilamine to mast cells was examined. Pyrilamine bound rapidly, in a saturable and reversible fashion, and with increased binding at 4°C as compared with 21°C and 37°C. [³H] Pyrilamine binding was displaced by H-1 antagonists (tripelnnamine > yrilamine ≧ iphenhydramine) > histamine > the H-2 antagonist, cimetidine. H-1 agonists displaced pyrilamine binding less efficiently than histamine but better than H-2 agonists. Rat mast cells have a single homogeneous population of low affinity (K_D = 222 ± 33 nM) H-1 receptors with a Bmax of 9.7 ± 2.3 pm/10⁶ mast cells and 5.4 ± 0.92 × 10⁶ binding sites per mast cell. Thus, the mast cell has an H-1 type histamine receptor which is probably involved in histamine-induced cyclic AMP increases.     

Internalization, recycling, and redistribution of vasopressin receptors in rat hepatocytes

Three hours after isolation, cultured hepatocytes have approximately 150,000 surface vasopressin receptors/cell, and these exhibit a Kd for 125I-vasopressin of 6 nM based on calculation of Koff/Kon, or a Kd of 9.5 nM based on Scatchard plot analysis. After the binding of 125I-vasopressin to its receptor on the hepatocyte surface, this complex is internalized with a t1/2 of 3-6 min. Following this internalization, the number of vasopressin receptors on the cell surface is restored both in vitro and in the isolated perfused liver with a t1/2 of 8-10 min. This restoration is blocked in vitro by incubation of the hepatocytes at 18 degrees C, but not by cycloheximide, suggesting that internalized vasopressin receptors recycle back to the cell surface. Prolonged incubation of hepatocytes with vasopressin results in the loss of greater than 75% of the vasopressin surface binding at concentrations of vasopressin approximately equivalent to its Kd. The binding of vasopressin to cultured hepatocytes 3-5 h after isolation resembles binding to the isolated perfused whole liver with respect to receptor dynamics. During culture for 48 h, however, we observe a progressive loss of hepatocyte surface vasopressin receptors. Concomitant with this reduction in surface receptors with time in culture, there appears to be a marked elevation in intracellular receptors.     

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.     

Kinetic analysis of cytokine‐mediated receptor assembly using engineered FC heterodimers

A method for analyzing ligand–receptor binding kinetics is described, which is based on an engineered FC domain (FChk) that forms a covalent heterodimer. To validate the system, the type I IFN receptors (IFNAR1 and IFNAR2) were expressed as IFNAR1‐FChk, IFNAR2‐FCkh, and IFNAR1/IFNAR2‐FChk fusion proteins. Surface plasmon resonance (SPR) analysis of binary IFNα2a/IFNAR interactions confirmed prior affinity measurements, while the affinity of the IFNα2a/IFNAR1/IFNAR2‐FChk interaction reproduced the affinity of IFNα2a binding to living cells. In cellular assays, IFNAR1/IFNAR2‐FChk potently neutralized IFNα2a bioactivity with an inhibitory concentration equivalent to the K_D measured by SPR. These studies suggest that FChk provides a simple reagent to evaluate the binding kinetics of multiple ligand–receptor signaling systems that control cell growth, development, and immunity.     

Receptor-mediated endocytosis of interleukin 2 in a human tumor T cell line. Degradation of interleukin 2 and evidence for the absence of recycling of interleukin receptors

We have previously described a human tumor T cell line, IARC 301, which constitutively expresses biologically functional interleukin 2 (IL2) receptors. The fate of IL2 after binding to surface high affinity receptors was investigated. After a few minutes, IL2 is internalized at 37 degrees C and is subsequently degraded and released in the medium. The half-life for surface high affinity IL2 receptors, as measured in the presence of cycloheximide, is about 1 h and does not depend upon the presence of IL2. After trypsin digestion of cell surface high affinity receptors in the absence of protein biosynthesis, we could not detect any receptor reappearance on the cell membrane, whether IL2 was added or not. Taken together these results show that IL2 receptors are constantly internalized in these cells in the presence or absence of IL2 and that they do not recycle to the plasma membrane after receptor-mediated endocytosis.     

Biochemical properties of the 1 alpha, 25-dihydroxyvitamin D3 cytoplasmic receptors from human and chick parathyroid glands

Cytoplasmic receptors for 1α, 25-dihydroxyvitamin D₃ from human parathyroid adenoma tissue and rachitic chick parathyroid glands have been characterized with regard to a number of physical, chemical, and ligand binding properties. Both receptors are 3.6–3.7 S proteins with molecular weights of approximately 75,000 and Stoke's molecular radii of 36 Å. It was found that the receptors possess a cysteine residue in or near the 1α, 25-dihydroxyvitamin D₃ binding site which is critical for ligand binding activity. The receptors both have equilibrium dissociation constants for 1α, 25-dihydroxyvitamin D₃ in the range of 2 to 5 × 10^?10m at 4 °C and second-order association rate constants for their seco-steroid ligand of 1 × 10⁷, m^?1 min^?1 (0 °C). The dissociation rate constants were found to be 5.3 × 10^?4 min^?1 (4 °C) for the human receptor and 1.3 × 10^?5 min^?1 (4 °C) for the chick receptor. The great deal of similarity which exists between the cytoplasmic 1α, 25-dihydroxyvitamin D₃ receptors from avian and mammalian parathyroid glands suggests a homologous function for these molecules in the two tissues.     

Binding of bovine parathyroid hormone to surface receptors of cultured B-lymphocytes

Binding of parathyroid hormone onto B-lymphocytes is detected by the utilization of the labelled antibody membrane assay. The amount of parathyroid hormone bound to the receptor sites was depending on the quantity of cells in the incubation milieu. Each cell line showed typical characteristics in time course of parathyroid hormone binding and maximal receptor capacity. Fragmentation of intact parathyroid hormone, also varying with the cell line tested, was very rapid, even at 24°C. Within 20 min most of the cell lines destroyed 50% of the native hormone in the incubation mixture, indicating a fragmentation rate of up to 2.25 ng/min at 37°C. B_max and K_D for the different lymphocytes was 5.3–19 · 10¹¹ M and 1.8–18.5 · 10¹¹ M, respectively. These values are in the range of reported plasma concentrations and may therefore represent more physiological values for the capacity and affinity of membrane receptors.     

Evidence for reutilization of surface receptors for alpha-macroglobulin.protease complexes in rabbit alveolar macrophages

Rabbit alveolar macrophages internalize α-macroglobulin ¹²⁵I-trypsin complexes subsequent to binding of complexes to high affinity surface receptors. Cells were capable of accumulating a 5–10 fold greater amount of αM · ¹²⁵I-T at 37°C than at 0°C. At 0°C cell-bound αM · ¹²⁵I-T was bound solely to surface receptors, whereas at 37°C the majority (85%) of cell-bound radioactivity was intracellular. The temperature-dependent accumulation of αM · ¹²⁵I-T did not reflect a change in surface receptor number or ligand-receptor affinity. Rather, the greater rate of uptake reflected continued internalization of αM · ¹²⁵I-T complexes. At 37°C cells took up 5–9 fmole αMT per μg cell protein per hr, whereas binding to surface receptors accounted for 0.5–0.7 fmole per μg cell protein. Once bound to surface receptors internalized αM · ¹²⁵I-T was localized in lysosomes, where it was degraded at a rate of 35–45% per hr. Following binding of αM · T to receptors at 37°C, but not at 0°C, unoccupied receptors could be found on the cell surface. Using cycloheximide to probe receptor turnover, I calculated that receptors were replenished at a rate of 15% per hr. Cells incubated in the presence of cycloheximide exhibited unaltered ligand uptake and catabolism for hours. Thus the reappearance of receptor activity during ligand uptake was not primarily due to de novo receptor synthesis. The rate of ligand uptake was a function of the number of surface receptors. Measurement of αM¹²⁵I-T binding to subcellular fractions did not reveal the presence of any intracellular reservoir of receptors. These observations are consistent with the hypothesis that continued ligand uptake reflects receptor reutilization.     

Benzodiazepine receptors: temperature dependence of [3H]flunitrazepam binding.

The characteristics of [³H]flunitrazepam binding to brain specific benzodiazepine receptors were determined at varying temperatures. The rates at which [³H]flunitrazepam associated with and dissociated from benzodiazepine receptors increased with increasing temperatures. The dissociation constant (K_D) also increased with increases in temperature. The (K_D) determined by Scatchard analyses of saturation isotherms showed a similar change with changes in temperature. The maximal binding capacity (Bmax) did not change with changes in temperature. The inhibitory constants of several benzodiazepines to inhibit [³H]flunitrazepam binding to brain were also higher at 37°C than at 0°C, suggesting that the binding affinity of all benzodiazepines to brain benzodiazepine receptors is lower at 37°C than at 0°C. Van't Hoff analysis of [³H]flunitrazepam binding to brain at different temperatures reveals two linear components to this relationship.     

A novel endocytic recycling signal distinguishes biological responses of Trk neurotrophin receptors

Endocytic trafficking of signaling receptors to alternate intracellular pathways has been shown to lead to diverse biological consequences. In this study, we report that two neurotrophin receptors (tropomyosin-related kinase TrkA and TrkB) traverse divergent endocytic pathways after binding to their respective ligands (nerve growth factor and brain-derived neurotrophic factor). We provide evidence that TrkA receptors in neurosecretory cells and neurons predominantly recycle back to the cell surface in a ligand-dependent manner. We have identified a specific sequence in the TrkA juxtamembrane region, which is distinct from that in TrkB receptors, and is both necessary and sufficient for rapid recycling of internalized receptors. Conversely, TrkB receptors are predominantly sorted to the degradative pathway. Transplantation of the TrkA recycling sequence into TrkB receptors reroutes the TrkB receptor to the recycling pathway. Finally, we link these divergent trafficking pathways to alternate biological responses. On prolonged neurotrophin treatment, TrkA receptors produce prolonged activation of phosphatidylinositol 3-kinase/Akt signaling as well as survival responses, compared with TrkB receptors. These results indicate that TrkA receptors, which predominantly recycle in signal-dependent manner, have unique biological properties dictated by its specific endocytic trafficking itinerary.     

Binding, internalization, and intracellular processing of proteins interacting with recycling receptors. A kinetic analysis

We measured time-dependent concentration changes of human interferon-alpha 2a (IFN) and human tumor necrosis factor-alpha (TNF) bound at the plasma membrane and internalized by human lung alveolar carcinoma A549 cells in the presence of excess free ligand. Concentration changes for these two ligands were substantially different. We modified our compartmental kinetic model encompassing receptor synthesis and receptor loss (Myers, A. C., Kovach, J. S., and Vuk-Pavlovi?, S. (1987) J. Biol. Chem. 262, 6494-6499) to include receptor recycling. We solved analytically the equations of three variants of the model of receptor recycling. All parameters (rate constants) were identifiable when the data sets consisted of time-resolved concentrations of IFN and TNF at the cell surface and internalized by cells. By least squares fitting we derived the best fit values for the first order rate constants for internalization of the ligand-receptor complex, receptor recycling, turnover of free receptors, elimination of the ligand from cells, and the rate of insertion of free receptors into the membrane. The best fit to data for interactions of cells with IFN was obtained without inclusion of the term for recycling of receptors to the membrane. The simplest model including receptor recycling was necessary and sufficient for the fit to the respective data for TNF. These results demonstrate that the contribution of receptor recycling to the metabolism of the ligand and the receptor can be quantitated by compartmental modeling. Receptor recycling does not contribute to the kinetics of Type I IFN receptor in A549 cells. In contrast, recycling contributes significantly to endocytosis mediated by the TNF receptor.     

Inhibitors of phosphoinositide 3-kinase cause defects in the postendocytic sorting of beta2-adrenergic receptors

Phosphatidylinositol 3-kinase inhibitors have been shown to affect endocytosis or subsequent intracellular sorting in various receptor systems. Agonist-activated beta(2)-adrenergic receptors undergo desensitization by mechanisms that include the phosphorylation, endocytosis and degradation of receptors. Following endocytosis, most internalized receptors are sorted to the cell surface, but some proportion is sorted to lysosomes for degradation. It is not known what governs the ratio of receptors that recycle versus receptors that undergo degradation. To determine if phosphatidylinositol 3-kinases regulate beta(2)-adrenergic receptor trafficking, HEK293 cells stably expressing these receptors were treated with the phosphatidylinositol 3-kinase inhibitors LY294002 or wortmannin. We then studied agonist-induced receptor endocytosis and postendocytic sorting, including recycling and degradation of the internalized receptors. Both inhibitors amplified the internalization of receptors after exposure to the beta-agonist isoproterenol, which was attributable to the sorting of a significant fraction of receptors to an intracellular compartment from which receptor recycling did not occur. The initial rate of beta(2)-adrenergic receptor endocytosis and the default rate of receptor recycling were not significantly altered. During prolonged exposure to agonist, LY294002 slowed the degradation rate of beta(2)-adrenergic receptors and caused the accumulation of receptors within rab7-positive vesicles. These results suggest that phosphatidylinositol 3-kinase inhibitors (1) cause a misrouting of beta(2)-adrenergic receptors into vesicles that are neither able to efficiently recycle to the surface nor sort to lysosomes, and (2) delays the movement of receptors from late endosomes to lysosomes.     

Post-endocytic fates of delta-opioid receptor are regulated by GRK2-mediated receptor phosphorylation and distinct beta-arrestin isoforms

Once internalized, some G protein-coupled receptors (GPCRs) can recycle back to the cell surface, while some of them are delivered to lysosomes for degradation. Because recycling and degradation represent two opposing receptor fates, understanding the mechanisms that determine post-endocytic fate of GPCRs is of great importance. Our recent work has verified that agonist-induced internalization of delta-opioid receptor (DOR) employs both phosphorylation-dependent and -independent mechanisms in HEK293 cells. To investigate whether these two internalization mechanisms work differently in receptor regulation, we monitored receptor post-endocytic fates using flow cytometry, surface receptor biotinylation and radioligand binding assays. Results showed that the internalized wild type DOR could either recycle to the cell surface or be degraded. Mutant DOR M4/5/6, which lacks all three G protein-coupled receptor kinase 2 (GRK2) phosphorylation sites, could also internalize upon agonist challenge although in a reduced level as compared with the wild type counterpart. However, the internalized mutant DOR could not recycle back to the cell surface and all mutant DOR was degraded after internalization. Inhibition of GRK2 expression by GRK2 RNAi also strongly attenuated recycling of DOR. Furthermore, overexpression of GRK2, which significantly increased receptor phosphorylation and internalization, also targeted more internalized receptors to the recycling pathway. These data suggest that GRK2-catalyzed receptor phosphorylation is critically involved in DOR internalization and recycling, and the phosphorylation-independent internalization leads to receptor degradation. Data obtained from beta-arrestin1 and beta-arrestin2 RNAi experiments indicated that both beta-arrestin1 and beta-arrestin2 participate in phosphorylation-dependent internalization and the subsequent recycling of DOR. However, phosphorylation-independent internalization and degradation of DOR were strongly blocked by beta-arrestin2 RNAi, but not beta-arrestin1 RNAi. Taken together, these data demonstrate for the first time that GRK2 phosphorylation-dependent internalization mediated by both beta-arrestin1 and beta-arrestin2 leads DOR to recycle, whereas GRK2-independent internalization mediated by beta-arrestin2 alone leads to receptor degradation. Thus, the post-endocytic fate of internalized DOR can be regulated by GRK2-catalyzed receptor phosphorylation as well as distinct beta-arrestin isoforms.     

Phosphorylation and recycling kinetics of G protein-coupled receptors

The rate of ligand-induced phosphorylation of the V2 and V1a vasopressin receptors was characterized in HEK 293 cells. Both receptors were phosphorylated predominantly by GRKs, and the V1a receptor was also phosphorylated by protein kinase C regardless of the presence or absence of ligand. Phosphorylation of the V1aR catalyzed by GRKs reached maximal values at the shortest measured time: 15 seconds, and decayed rapidly with a t1/2 of 6 min in the continuous presence of AVP. In agreement with the hypothesis that dephosphorylation must precede receptor recycling to the cell surface, the V1aR returned rapidly to the cell surface after removal of the hormone from the medium. Phosphate incorporation into the V2R proceeded at a slower pace, and the internalized phosphorylated receptor failed to recycle to the cell surface and retained its phosphate for a long time in the presence or absence of ligand. A single mutation in the carboxy terminus of the V2R accelerated de-phosphorylation of the protein and conferred recycling properties to the V2R. These experiments provided molecular evidence for the hypothesis that internalization is required for de-phosphorylation and recycling of reactivated G protein coupled receptors to the cell surface.     

Dimerization of internalized epidermal growth factor receptors.

Binding of epidermal growth factor (EGF) to cell surface EGF receptors initiates the formation of the receptor homodimers that can be detected by covalent cross-linking in intact cells or in detergent-solubilized cell extracts. Low pH dissociation of EGF from surface receptors results in immediate monomerization of receptor dimers. Using chemical cross-linking during mild permeabilization or cell solubilization, we have detected dimers of internalized EGF receptors in human carcinoma A-431 cells and transfected NIH 3T3 cells that express human EGF receptors. The percentage of internalized cross-linked receptor dimers was similar to that observed for surface EGF receptors. Furthermore, at the time of maximal accumulation of EGF-receptor complexes within the endosomal compartment (10-15 min of incubation at 37 degrees C), both the dimeric and monomeric forms of the EGF receptor are tyrosine-phosphorylated to the same extent as surface dimer and monomer species. In transfected NIH 3T3 cells, the level of dimerized and internalized kinase-negative EGF receptors was not different from that observed for wild-type receptors. These data suggest that for some time after internalization EGF does not dissociate from its receptor and indicate that a receptor conformation is preserved intracellularly that allows maintenance of receptor-receptor interactions and tyrosine kinase activity.     

Endosomal location of dopamine receptors in neuronal cell cytoplasm

Five subtypes of dopamine receptor exist in two subfamilies: two D₁-like (D₁ and D₅) and three D₂-like (D₂, D₃ and D₄). We produced novel monoclonal antibodies against all three D₂-like receptors and used them to localize receptors in Ntera-2 (NT-2) cells, the human neuronal precursor cell line. Most of the immunostaining for all three receptors colocalized with mannose-6-phosphate receptor, a marker for late endosomes formed by internalization of the plasma membrane. This result was obtained with antibodies against three different epitopes on the D₃ receptor, to rule out the possibility of cross-reaction with another protein, and controls without primary antibody or in the presence of competitor antigen were completely negative. In rat cerebral cortex and hippocampus, some of the dopamine receptor staining was found in similar structures in neuronal cell cytoplasm. Only some of the neurons were positive for dopamine receptors and the pattern was consistent with previously-reported patterns of innervation by dopamine-producing neurons. Endosomal dopamine receptors may provide a useful method for identifying cell bodies of dopamine-responsive neurons to complement methods that detect only active receptors in the neuronal cell membrane.     

Effect of membrane flow on the capture of receptors by coated pits. Theoretical results.

Coated pits trap cell surface receptors and mediate their internalization. Once internalized, many receptors recycle back to the cell surface. When recycled receptors are inserted into the plasma membrane, they move until they are again trapped in coated pits. The mechanisms for moving receptors from their insertion sites to coated pits are unknown. Unaided diffusion as the transport mechanism is consistent with the observed kinetics of receptor recycling. Another candidate for the transport mechanism is convection. For receptors that recycle to random positions on the cell surface, or to restricted regions about coated pits, we assess the importance of convective flow in the transport of receptors to coated pits. First we consider local flows set up by the formation of coated pits and their transformation into coated vesicles. As coated pits form and round into coated vesicles, surrounding membrane is drawn inward, creating flows directed toward the coated pit centers. We show that unless the lifetime of a coated pit is very short, 10 s or less, such local flows have a negligible effect on the time it takes receptors to reach coated pits. We also show that they are unlikely to be the mechanism that keeps receptors that have reached coated pits trapped within coated pits until they are internalized. Finally we calculate the mean time tau for a diffusing receptor to reach a coated pit in the presence of membrane flow that is constant in magnitude and direction, as may occur on moving cells. We show that for typical membrane flow velocities, tau can be reduced significantly from its value in the absence of flow. For example, a velocity v = 2.8 micron/min cuts the mean transport time in half.     

Epidermal growth factor receptors

EGF-Rs are cell membrane glycoproteins of wide distribution. They have not yet been fully characterized or purified but are probably molecules of 170-190,000 mol. wt. in most cells. The growth factor EGF binds and will saturate cell surface receptors with a KA of about 5 X 10(9) M-1 although a receptor class with an affinity in excess of 10(10) M-1 has been detected in some cells. The number of receptors on a cell does not determine the level of its response. Some cell types have receptors which bind EGF, but with no mitogenic response. The ways in which receptor affinity and/or number is modulated are described. This and other evidence is reviewed in a search for a suitable model of a mechanism of action on the cell, which best fits the current data. There is ample evidence that EGF binds to the receptor; that ligand-receptor complexes cluster or aggregate; and then are internalized and degraded, but evidence for a direct connection between internalization and the subsequent mitogenic response is lacking. Good correlations between internalization and mitogenic responses have been observed and developed into a theory of endocytic activation, but there is a body of evidence which cannot be accommodated by this theory. Instead, an alternative model is suggested.