EGF receptor residues leu(679), leu(680) mediate selective sorting of ligand-receptor complexes in early endosomal compartments

Dileucine-based motifs have been shown to regulate endosomal sorting of a number of membrane proteins. Previously, we have shown that the dileucine motif Leu(679), Leu(680) in the juxtamembrane domain of the human epidermal growth factor receptor is involved in the endosome-to-lysosome transport of ligand-receptor complexes. Substitution of alanine residues for Leu(679), Leu(680) led to a reduction in ligand-induced receptor degradation without affecting internalization. In the current study, we have further characterized ligand-dependent intracellular sorting of EGF receptors containing a L679A, L680A. Immunocytochemical studies reveal that although mutant receptors redistribute from the cell surface to transferrin receptor-positive endocytic vesicles similar to wild-type following ligand stimulation, their accumulation in Lamp-1-positive late endosomes/lysosomes is retarded compared to wild-type. Kinetic analysis of (125)I-EGF trafficking shows that reduced accumulation of internalized mutant receptors in Lamp-1-positive vesicles is due to rapid recycling of ligand-receptor complexes from early endocytic compartments. In addition, the fraction of intracellular (125)I-EGF that is transported to late endocytic compartments in cells with mutant receptors is not as efficiently degraded as it is in cells with wild-type receptors. Furthermore, wild-type receptors in endocytic vesicles isolated by Percoll gradient fractionation are more resistant to in vitro digestion with proteinase K than mutant receptors. We propose that mutant receptors interact inefficiently with lysosomal sorting machinery, leading to their increased recycling. Our results are consistent with a model in which the Leu(679), Leu(680) signal facilitates sequestration of ligand-receptor complexes into internal vesicles of multivesicular endosome-to-lysosome transport intermediates.     

The capping of receptors for the epidermal growth factor and the participation of the cytoskeleton in this process in A-431 cells

Epidermal growth factor (EGF) receptor capping results from the interaction between the receptors and polyvalent ligands in A-431 cells examined in suspension at 22 degrees C. Colocalization of actin and spectrin with the ligand-receptor complexes during the redistribution was shown using double immunofluorescence. The obtained data show that the cortical microfilaments are involved in capping. EGF receptors become associated with the Triton-insoluble cytoskeleton as a consequence of ligand binding. EGF-receptor capping is not sensitive to the action of cytochalasin B. Capping in A-431 cells is discussed as a new model for studying the redistribution of the ligand-receptor complex.     

Model of competitive binding of vascular endothelial growth factor and placental growth factor to VEGF receptors on endothelial cells

Placental growth factor (PlGF) competes with vascular endothelial growth factor (VEGF) for binding to VEGF receptor (VEGFR)-1 but does not bind VEGFR2. Experiments show that PlGF can augment the response to VEGF in pathological angiogenesis and in models of endothelial cell survival, migration, and proliferation. This synergy has been hypothesized to be due to a combination of the following: signaling by PlGF through VEGFR1 and displacement of VEGF from VEGFR1 to VEGFR2 by PlGF, causing increased signaling through VEGFR2. In this study, the relative contribution of PlGF-induced VEGF displacement to the synergy is quantified using a mathematical model of ligand-receptor binding to examine the effect on ligand-receptor complex formation of VEGF and PlGF acting together. Parameters specific to the VEGF-PlGF system are used based on existing data. The model is used to simulate in silico a specific in vitro experiment in which VEGF-PlGF synergy is observed. We show that, whereas a significant change in the formation of endothelial surface growth factor-VEGFR1 complexes is predicted in the presence of PlGF, the increase in the number of VEGFR2-containing signaling complexes is less significant; these results were shown to be robust to significant variation in the kinetic parameters of the model. Synergistic effects observed in that experiment thus appear unlikely to be due to VEGF displacement but to a shift from VEGF-VEGFR1 to PlGF-VEGFR1 complexes and an increase in total VEGFR1 complexes. These results suggest that VEGFR1 signaling can be functional in adult-derived endothelial cells.     

Selection against demographic stochasticity in age-structured populations

It has been shown that differences in fecundity variance can influence the probability of invasion of a genotype in a population; i.e., a genotype with lower variance in offspring number can be favored in finite populations even if it has a somewhat lower mean fitness than a competitor. In this article, Gillespie's results are extended to population genetic systems with explicit age structure, where the demographic variance (variance in growth rate) calculated in the work of Engen and colleagues is used as a generalization of "variance in offspring number" to predict the interaction between deterministic and random forces driving change in allele frequency. By calculating the variance from the life-history parameters, it is shown that selection against variance in the growth rate will favor a genotypes with lower stochasticity in age-specific survival and fertility rates. A diffusion approximation for selection and drift in a population with two genotypes with different life-history matrices (and therefore different mean growth rates and demographic variances) is derived and shown to be consistent with individual-based simulations. It is also argued that for finite populations, perturbation analyses of both the mean and the variance in growth rate may be necessary to determine the sensitivity of fitness to changes in the life-history parameters.     

Analysis of some "insoluble" problems of determining the binding parameters of ligand-receptor interaction and methods of their solving

Some problems of the estimation of the parameters of ligand-receptor interaction (affinity, rate constants, valency, etc.) were considered. It was demonstrated that not only the Scatchard plot but also Klotz plot could be used for determining the parameters of ligand-receptor interaction for two types of binding sites of different affinity. A new approach and new coordinate systems for the estimation of the parameters of ligand-receptor interaction were suggested. It was shown that for the estimation of the affinity of putative monovalent antibodies by ELISA various equations, which are more precise and convenient than the Friguet et al. equations, could be obtained by the transformation of mass action law equation. The problem solution for the estimation by ELISA the affinity of two types of bivalent antibodies with different affinity and their concentrations for the case of the mixture of these antibodies was also suggested. The application of the proposed coordinate of dilution allows to solve the problem of determination of the parameters of ligand-receptor interaction (including antigen-antibody system) for the pre-existing ligand-receptor mixture without their preliminary separation and purification. This approach is especially important for the cases when the receptor is not stable enough to be isolated in the intact form from this mixture. It was shown that the well-known phenomenon of the prozone often observed under the titration of serum antibodies by the method of agglutination may get a mathematical explanation. Analytical solution of the problem of determining the velocity constant and the amount of the end product of the first order irreversible and reversible reaction kinetics was suggested, despite the fact that the process is described by the system of irrational equations. Mehods of asymptotic solution of transcendental irrational equations which describe the dynamics of reactions which mechanisms are subject to the so-called heterogeneous, successive, or competitive models have been considered. These methods permit the finding of the reaction rate constants and the amount of the end product, if the kinetics of the transformation of either initial, or end product of the reaction is known.     

Effect of monensin on receptor recycling during continuous endocytosis of asialoorosomucoid

The binding of asialoglycoproteins to their liver cell receptor results in internalization of the ligand-receptor complex. These complexes rapidly appear in intracellular compartments termed endosomes whose acidification results in ligand-receptor dissociation. Ligand and receptor subsequently segregate: ligand is transported to lysosomes and is degraded while receptor recycles to the cell surface. The proton ionophore monensin prevents acidification of endosomes and reversibly inhibits this acid-dependent dissociation of ligand from receptor. The present study determined the effect of monensin treatment of short-term cultured rat hepatocytes on cell-surface-receptor content, determined both by their binding activity and immunologically, following continuous endocytosis of asialoorosomucoid. Inclusion of 5 microM monensin in the incubation medium reduced the number of immunologically detectable cell-surface receptors by 20% in the absence of ligand. During continuous endocytosis of asialoorosomucoid, inclusion of monensin resulted in a 30-40% reduction of cell-surface receptor detectable either by ligand binding or immunologically. These results suggest that the reduced liver-cell-surface content of receptor in monensin is due to intracellular trapping of ligand-receptor complexes. The reduction of surface receptor during monensin incubation in the absence of ligand suggests that "constitutive recycling" of plasma membrane components also requires intracellular acidification.     

Dependence of the redistribution of ligand-receptor complexes in cultured cells on the type of growth and on the cell cycle phase

The process of active redistribution of ligand-receptor complexes on the surface of both suspension (LS) and adapted for growing in monolayer (LMS) sublines of mouse fibroblasts L was studied. The binding of ligands to its specific receptors on the surface of the LS cells induced an accumulation of ligand-receptor complexes on one pole of a cell with the formation of a typical cap. Under the same conditions in the LSM cells the cleaning of processes and lamello-plasma surfaces from the ligand-receptor complexes was registered. The binding of ligands to the surface of the LSM cells, detached with EDTA, induced the same capping process, as in the case of the LS cells. No differences were found in the redistribution capacity of the ligand-receptor complexes in synchronized cultures of the LS and LSM cells in the G1 and S phase of the cycle. But such a redistribution was not registered on the surface of cells in metaphase and anaphase. The accumulation of ligand-receptor complexes was found in the region of cleavage between daughter cells in telophase. These results are in a good agreement with the well-known data on the changes in the cytoskeleton organization during transition from a monolayer to a suspension state and during mitosis.     

Interaction between electromagnetic fields and cells: microelectrophoretic effect on ligands and surface receptors

The aggregation between lectins and lymphocyte surface receptors can be affected strongly by a low-level electric field induced in the cell suspension by a time-varying magnetic field. One of the possible mechanisms is the microelectrophoretic effect due to the electric field, which influences the distance (in the mean square sense) between charged ligands and receptors when they are about to separate. On a purely theoretical basis, it is shown that, at low frequencies, an externally induced periodic electric field always decreases the mean lifetime of ligand-receptor complexes. As a consequence, the mitogenic gain obtained by lectin addition to cell suspension is decreased. These results suggest that such a mechanism, if effective, reduces the lectin mitogenic capability and offers a way of handling similar phenomena which have been described for other biological systems.     

Big gulps: specialized membrane domains for rapid receptor-mediated endocytosis

Eukaryotic cells are well known to use an elaborate, clathrin-based endocytic machinery to internalize ligand-receptor complexes. Although the number of components identified as being used during this essential process has increased substantially over the past decade, an appreciation for how receptor-mediated endocytosis is organized at a broader, cellular scale is still mostly undefined. Here, some new insights into how cells cluster and organize the endocytic machinery at defined cytoplasmic domains to increase the speed and efficiency of ligand-receptor internalization are presented.     

Receptor dynamics of closely related ligands: "fast'' and "slow'' interferons.

Two related human alpha interferons with 83% homology in their primary sequences show a similar specific activity on nonhuman cells, but a striking difference on human cells, on which alpha-1 shows 1-5% of the specific molar activity displayed by alpha-2. Both interferons were labelled with 125I, and their binding kinetics followed on growing cultures of the human Burkitt line Daudi. Binding of alpha-1 showed slower rates of association and faster rates of dissociation implying that differences in apparent binding affinity were responsible for the differences in specific molar activity. However, binding was shown to reach steady-state rather than an equilibrium, so differences in the dynamics of the ligand-receptor complexes may represent amplification of differences in the initial binding constant. alpha-2, but not alpha-1, induces a marked loss of binding sites leading to a high affinity steady-state binding. Inhibition of cell multiplication by both interferons depends on a continued stimulation by free ligands at steady-state. It is proposed that the differences in specific molar activity are, in the main, kinetic and cause alpha-1 and alpha-2 to behave respectively as "slow' and "fast' interferons.     

Association of ligand-receptor complexes with actin filaments in human neutrophils: a possible regulatory role for a G-protein

Most ligand-receptor interactions result in an immediate generation of various second messengers and a subsequent association of the ligand- receptor complex to the cytoskeleton. Depending on the receptor involved, this linkage to the cytoskeleton has been suggested to play a role in the termination of second messenger generation and/or the endocytic process whereby the ligand-receptor complex is internalized. We have studied how the binding of chemotactic peptide-receptor complexes to the cytoskeleton of human neutrophils is accomplished. As much as 76% of the tritiated formylmethionyl-leucyl-phenylalanine (fMet- Leu-[3H]Phe) specifically bound to intact cells, obtained by a 30-s stimulation with 20 nM fMet-Leu-[3H]Phe, still remained after Triton X- 100 extraction. Preincubating intact cells with dihydrocytochalasin B (dhCB) or washing the cytoskeletal preparation with a high concentration of potassium, reduced the binding of ligand-receptor complexes to the cytoskeleton by 46% or more. Inhibition of fMet-Leu- Phe-induced generation of second messengers by ADP-ribosylating the alpha-subunit of the receptor-coupled G-protein with pertussis toxin, did not reduce the binding of ligand-receptor complexes to the cytoskeleton. However, using guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) to prevent the dissociation of the fMet-Leu-Phe-associated G- protein within electrically permeabilized cells, led to a pronounced reduction (62%) of the binding between ligand-receptor complexes and the cytoskeleton. In summary, in human neutrophils the rapid association between chemotactic peptide-receptor complexes and the cytoskeleton is dependent on filamentous actin. This association is most likely regulated by the activation and dissociation of the fMet- Leu-Phe-associated G-protein.     

The complexity of complexes in signal transduction

Many activities of cells are controlled by cell-surface receptors, which in response to ligands, trigger intracellular signaling reactions that elicit cellular responses. A hallmark of these signaling reactions is the reversible nucleation of multicomponent complexes, which typically begin to assemble when ligand-receptor binding allows an enzyme, often a kinase, to create docking sites for signaling molecules through chemical modifications, such as tyrosine phosphorylation. One function of such docking sites is the co-localization of enzymes with their substrates, which can enhance both enzyme activity and specificity. The directed assembly of complexes can also influence the sensitivity of cellular responses to ligand-receptor binding kinetics and determine whether a cellular response is up- or downregulated in response to a ligand stimulus. The full functional implications of ligand-stimulated complex formation are difficult to discern intuitively. Complex formation is governed by conditional interactions among multivalent signaling molecules and influenced by quantitative properties of both the components in a system and the system itself. Even a simple list of the complexes that can potentially form in response to a ligand stimulus is problematic because of the number of ways signaling molecules can be modified and combined. Here, we review the role of multicomponent complexes in signal transduction and advocate the use of mathematical models that incorporate detail at the level of molecular domains to study this important aspect of cellular signaling.     

The dispersion of age differences between partners and the asymptotic dynamics of the HIV epidemic

In this paper, the effect of a change in the distribution of age differences between sexual partners on the dynamics of the HIV epidemic is studied. In a gender- and age-structured compartmental model, it is shown that if the variance of the distribution is small enough, an increase in this variance strongly increases the basic reproduction number. Moreover, if the variance is large enough, the mean age difference barely affects the basic reproduction number. We, therefore, conclude that the local stability of the disease-free equilibrium relies more on the variance than on the mean.     

The dispersion of age differences between partners and the asymptotic dynamics of the HIV epidemic

In this paper, the effect of a change in the distribution of age differences between sexual partners on the dynamics of the HIV epidemic is studied. In a gender- and age-structured compartmental model, it is shown that if the variance of the distribution is small enough, an increase in this variance strongly increases the basic reproduction number. Moreover, if the variance is large enough, the mean age difference barely affects the basic reproduction number. We, therefore, conclude that the local stability of the disease-free equilibrium relies more on the variance than on the mean.     

Two kinds of "recombination nodules" in Neurospora crassa

Two morphological types of recombination nodules, termed early and late, are recognized in Neurospora crassa. Eighty nuclei at different substages were used to determine numbers of nodules per nucleus, distribution of nodules along the nucleolus-organizing chromosome, and distribution of nodules among the two largest chromosomes. Early nodules appear at the synaptonemal complex at early zygotene and increase in number during zygotene until a dramatic reduction occurs at zygotene-pachytene transition. Thereafter early nodules are steadily eliminated until they disappear by diplotene. Late nodules are also present during zygotene. Their number doubles at the zygotene-pachytene transition and stays at this level until diplotene. The total number of nodules is rather constant through zygotene and pachytene. Distribution of bivalents with 0, 1, 2, etc. nodules follows a Poisson distribution at zygotene, but not at pachytene, where variance is less than the mean, indicating positive interference. Nodules are distributed nonrandomly along the nucleolus-organizer bivalent. The pattern differs slightly in nuclei of different origin. Nuclei with unusual synaptonemal complexes sustain normal levels of recombination by having the same amount of nodules as normal nuclei. In abnormal nuclei nodules are preferentially associated with normal segments. It is proposed that early nodules do not participate in any form of recombination but have a role in finding an appropriate site for a crossing-over event. Morphological change to the late type indicates that the site has been reached and the exchange event can be mediated by the late nodule.     

Consequences of ligand bivalency in interactions involving particulate receptors: equilibrium and kinetic studies with Sephadex-concanavalin A, butylagarose-phosphorylase b, and Fc receptor-IgG dimer interactions as model systems

Theoretical consideration is given to the interaction of a bivalent ligand with particulate receptor sites, not only from the viewpoint of quantitatively describing the binding behavior but also from that of the kinetics of ligand release upon infinite dilution of a receptor-ligand mixture. In the latter regard, a general expression is derived that describes the time dependence of the amount of ligand bound as a function of two rate constants for the stepwise dissociation of cross-linked ligand-receptor complex and a thermodynamic parameter expressing the initial ratio of singly linked to doubly linked ligand-receptor complexes. An experimental study of the interaction between Sephadex and concanavalin A is then used to illustrate application of this recommended theoretical approach for characterizing the binding behavior and dissociation kinetics of a bivalent ligand for a system in which all ligand-receptor interactions may be described by a single intrinsic association constant. Published results on the interaction of phosphorylase b with butylagarose are also shown to comply with this simplest model of the bivalent ligand hypothesis; but those for the interaction between immunoglobulin G (IgG) dimers and Fc receptors require modification of the model by incorporation of different intrinsic association constants for the successive binding of receptor sites to the bivalent ligand. These results emphasize the need to consider ligand bivalency as a potential phenomenon in studies of interactions between protein ligands and particulate receptors and illustrate procedures by which the effects of ligand bivalency may be identified and characterized.     

Ligand-regulated internalization, trafficking, and down-regulation of guanylyl cyclase/atrial natriuretic peptide receptor-A in human embryonic kidney 293 cells.

We examined the kinetics of internalization, trafficking, and down-regulation of recombinant guanylyl cyclase/natriuretic peptide receptor-A (NPRA) utilizing stably transfected 293 cells expressing a very high density of receptors. After atrial natriuretic peptide (ANP) binding to NPRA, ligand-receptor complexes are internalized, processed intracellularly, and sequestered into subcellular compartments, which provided an approach to examining directly the dynamics of metabolic turnover of NPRA in intact cells. The translocation of ligand-receptor complexes from cell surface to intracellular compartments seems to be linked to ANP-dependent down-regulation of NPRA. Using tryptic proteolysis of cell surface receptors, it was found that approximately 40-50% of internalized ligand-receptor complexes recycled back to the plasma membrane with an apparent t(12) = 8 min. The recycling of NPRA was blocked by the lysosomotropic agent chloroquine, the energy depleter dinitrophenol, and also by low temperature, suggesting that recycling of the receptor is an energy- and temperature-dependent process. Data suggest that approximately 70-80% of internalized (125)I-ANP is processed through a lysosomal degradative pathway; however, 20-25% of internalized ligand is released intact into the cell exterior through an alternative mechanism involving an chloroquine-insensitive pathway. It is implied that internalization and processing of bound ANP-NPRA complexes may play an important role in mediating the biological action of hormone and the receptor protein. In retrospect, this could occur at the level of receptor regulation or through the initiation of ANP mediated signals. It is envisioned that the endocytotic pathway of ligand-receptor complexes of ANP-NPRA would lead to termination and/or diminished responsiveness of ANP in target cells.     

Solubilization of the bombesin receptor from Swiss 3T3 cell membranes. Functional association to a guanine nucleotide regulatory protein

Bombesin and structurally related peptides including gastrin releasing peptide (GRP) are potent mitogens for Swiss 3T3 cells. Here we attempted to solubilize bombesin receptors under conditions in which the ligand (125I-labelled GRP) was prebound to the receptor prior to detergent extraction. We found that 125I-GRP-receptor complexes were solubilized from Swiss 3T3 cell membranes by using the detergents taurodeoxycholate or deoxycholate. These detergents promoted ligand-receptor solubilization in a dose-dependent manner. In contrast, a variety of other detergents including Triton X-100, octylglycoside, CHAPS, digitonin, cholic acid and n-dodecyl-beta-D-maltoside, were much less effective. Addition of guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) to ligand-receptor complexes isolated by gel filtration enhanced the rate of ligand dissociation in a concentration-dependent and nucleotide-specific manner. Our results demonstrate for the first time the successful solubilization of 125I-GRP-receptor complexes from Swiss 3T3 cell membranes and provide evidence for the physical association between the ligand-receptor complex and a guanine nucleotide binding protein(s).