Effect Of Receptor Density Research Articles

Triptorelin-functionalized PEG-coated biosynthesized gold nanoparticles: Effects of receptor-ligand interactions on adhesion to triple negative breast cancer cells.

This paper presents the results of an experimental and computational study of the adhesion of triptorelin-conjugated PEG-coated biosynthesized gold nanoparticles (GNP-PEG-TRP) to triple-negative breast cancer (TNBC) cells. The adhesion is studied at the nanoscale using a combination of atomic force microscopy (AFM) experiments and molecular dynamics (MD) simulations. The AFM measurements showed that the triptorelin-functionalized gold nanoparticles (GNP-TRP and GNP-PEG-TRP) have higher adhesion to triple-negative breast cancer cells (TNBC) than non-tumorigenic breast cells. The increased adhesion of GNP-TRP and GNP-PEG-TRP to TNBC is also attributed to the overexpression of LHRH receptors on the surfaces of both TNBC. Finally, the molecular dynamics model reveals insights into the effects of receptor density, molecular configuration, and receptor-ligand docking characteristics on the interactions of triptorelin-functionalized PEG-coated gold nanoparticles with TNBC. A three to nine-fold increase in the adhesion is predicted between triptorelin-functionalized PEG-coated gold nanoparticles and TNBC cells. The implications of the results are then discussed for the specific targeting of TNBC.

Effects of receptor density on the tactile perception of roughness: implications for neural mechanisms of texture perception

Aim of the study: The purpose of this study was to investigate the effects of receptor density in the glabrous skin of the hand on the perception of the roughness of a textured surface. Materials and methods: This was done by having observers make magnitude estimates of the perceived roughness of raised-dot surfaces at the fingertip, with its high receptor density, and the thenar eminence, with its much lower receptor density. Results: Judgments of perceived roughness averaged over the inter-dot spacings (0.8-5.9 mm) employed in the study did not differ significantly between the two sites, which suggested that roughness perception is not exclusively dependent upon a neural code involving variation in the activity levels of the nerve fibers of spatially distributed receptors, as is the case in spatial discrimination tasks such as spatial-gap detection, grove-orientation discrimination and letter recognition. This hypothesis was further supported by the finding that the elimination of temporal cues by preventing movement of the skin over the raised-dot surface drastically impaired judgments of perceived roughness at the thenar but had little effect on judgments of perceived roughness at the fingertip. Conclusion: These findings suggested that the neural code for perceived roughness at the fingertip is mediated primarily by spatial variation in the activity levels of spatially distributed receptors whereas the neural code for perceived roughness at the thenar is mediated primarily by temporal variation in the activity levels of individual receptors.

Evidence for an effect of receptor density on ligand occupancy and agonist EC50

Drug-receptor interaction theory predicts that proportional receptor occupancy is a function of ligand concentration as defined by a ligand-receptor affinity constant, and is independent of receptor density. However, we previously observed that the EC50 of 5-HT reduced as the density of 5-HT3 receptors increased, suggesting an effect of receptor density on occupancy. The current study was designed to maximise variability in experimentally observed currents and confirm this apparent contradiction prospectively. Xenopus oocytes were injected with RNA encoding 5-HT3A receptors under conditions designed to achieve varying receptor expression levels and 5-HT-evoked currents measured using two electrode voltage clamp. Results from 99 oocytes showed that as the maximal peak current increased from 0.05 µA to 12.1 µA there was a 3.7-fold reduction in EC50. Since occupancy and conductance are directly related in this system, this indicates that for a given concentration of 5-HT, proportional occupancy increases with increased receptor density. We conclude that normalising data masks this correlation, and can result in reduced accuracy of pharmacological measurements. We propose a mechanistic explanation for our observations.

PH regulation in early endosomes and interferon-inducible transmembrane proteins control avian retrovirus fusion

Enveloped viruses infect host cells by fusing their membranes with those of the host cell, a process mediated by viral glycoproteins upon binding to cognate host receptors or entering into acidic intracellular compartments. Whereas the effect of receptor density on viral infection has been well studied, the role of cell type-specific factors/processes, such as pH regulation, has not been characterized in sufficient detail. Here, we examined the effects of cell-extrinsic factors (buffer environment) and cell-intrinsic factors (interferon-inducible transmembrane proteins, IFITMs), on the pH regulation in early endosomes and on the efficiency of acid-dependent fusion of the avian sarcoma and leukosis virus (ASLV), with endosomes. First, we found that a modest elevation of external pH can raise the pH in early endosomes in a cell type-dependent manner and thereby delay the acid-induced fusion of endocytosed ASLV. Second, we observed a cell type-dependent delay between the low pH-dependent and temperature-dependent steps of viral fusion, consistent with the delayed enlargement of the fusion pore. Third, ectopic expression of IFITMs, known to potently block influenza virus fusion with late compartments, was found to only partially inhibit ASLV fusion with early endosomes. Interestingly, IFITM expression promoted virus uptake and the acidification of endosomal compartments, resulting in an accelerated fusion rate when driven by the glycosylphosphatidylinositol-anchored, but not by the transmembrane isoform of the ASLV receptor. Collectively, these results highlight the role of cell-extrinsic and cell-intrinsic factors in regulating the efficiency and kinetics of virus entry and fusion with target cells.

Binding of GTPγ[35S] is regulated by GDP and receptor activation. Studies with the nociceptin/orphanin FQ receptor

Background and purpose: We have examined the effects of ligand efficacy and receptor density on the binding of guanosine 5′‐[γ‐thio]triphosphate (GTPγS) and GDP to the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP)‐coupled G‐proteins.Experimental approach: In GTPγ[35S] binding experiments, using stable (CHOhNOP) and inducible (CHOINDhNOP) recombinant human and rat NOP we have measured: (i) ligand‐specific GDP requirements; (ii) the effects of receptor density on guanine nucleotide affinity/capacity; and (iii) the effect of ligand efficacy on GTPγS association kinetics.Key results: GTPγS competition curves were shallow and modelled by high‐ and low‐affinity components that were relatively consistent between cell types and tissue preparations. In the presence of 1 µM N/OFQ a high‐affinity GDP binding site was also present, but the fraction of total binding was reduced. In an efficacy‐dependent manner, the partial agonists [F/G]N/OFQ(1‐13)NH2 ([Phe1ψ(CH2‐NH)Gly2]‐nociceptin(1‐13)NH2) and naloxone benzoylhydrazone both reduced the fraction of high‐affinity sites for GDP (relative to basal). While the pIC50 for high‐affinity GDP binding site did not decrease in the presence of 1 µM N/OFQ, N/OFQ produced a significant reduction in pIC50 for the low‐affinity site. Agonist‐mediated decrease in affinity for GDP binding was efficacy‐dependent. GDP displayed three affinities: high, conserved in the presence and absence of ligand; intermediate, present as a low fraction under basal conditions; low (efficacy‐dependent), present during receptor activation representing the majority of binding.Conclusions and implications: The affinity of GTPγ[35S] was regulated by GDP and receptor activation caused increased binding of GTPγ[35S] through a reduction in GDP affinity.

Monte Carlo Simulation of Buffered Diffusion into and out of a Model Synapse

Buffered diffusion occurs when ligands enter or leave a restricted space, such as a chemical synapse, containing a high density of binding sites. This study used Monte Carlo simulations to determine the time and spatial dependences of buffered diffusion without a priori assumptions about kinetics. The synapse was modeled as a box with receptors on one inner face. The exterior was clamped to some ligand concentration and ligands diffused through two sides. Onset and recovery simulations were carried out and the effects of receptor density, ligand properties and synapse geometry were investigated. This study determined equilibration times for binding and the spatial gradient of unliganded receptors. Onset was characterized by a high spatial gradient; equilibration was limited by the time needed for sufficient ligands to enter the synapse. Recovery showed a low spatial gradient with receptor equilibration limited by ligand rebinding. Decreasing ligand association rate or increasing ligand diffusion coefficient reduced the role of buffered diffusion and decreased the spatial gradient. Simulations with irreversible ligands showed larger, persistent spatial gradients. These simulations identify characteristics that can be used to test whether a synaptic process is governed by buffered diffusion. They also indicate that fundamental differences in synapse function may occur with irreversible ligands.

Glycinergic Transmission Shaped by the Corelease of GABA in a Mammalian Auditory Synapse

The firing pattern of neurons is shaped by the convergence of excitation and inhibition, each with finely tuned magnitude and duration. In an auditory brainstem nucleus, glycinergic inhibition features fast decay kinetics, the mechanism of which is unknown. By applying glycine to native or recombinant glycine receptors, we show that response decay times are accelerated by addition of GABA, a weak partial agonist of glycine receptors. Systematic variation in agonist exposure time revealed that fast synaptic time course may be achieved with submillisecond exposures to mixtures of glycine and GABA at physiological concentrations. Accordingly, presynaptic terminals generally contained both transmitters, and depleting terminals of GABA slowed glycinergic synaptic currents. Thus, coreleased GABA accelerates glycinergic transmission by acting directly on glycine receptors, narrowing the time window for effective inhibition. Packaging both weak and strong agonists in vesicles may be a general means by which presynaptic neurons regulate the duration of postsynaptic responses.

Receptor density dictates the behavior of a subset of steroid ligands in glucocorticoid receptor-mediated transrepression

By co-expressing glucocorticoid receptor (GR) and transcriptional reporter systems in GR-deficient Cos-7 cells, we profiled potency and efficacy of a panel of GR ligands as a function of GR expression levels (density). Our results show that potency and efficacy for GR full agonists, such as dexamethasone, in these transrepression assays are affected by receptor density. Intriguingly, receptor density dramatically influenced the behavior of the GR antagonist RU486 or the GR agonist medroxyprogesterone acetate (MPA). At high receptor density, both MPA and RU486 behaved as full agonists in transrepression: reducing GR density, however, resulted in conversion of these ligands from full agonist to full antagonists. In contrast, varying GR density could not convert cortisol and budesonide from GR agonists to antagonists. These results have clearly demonstrated, for the first time, an effect of receptor density on the agonist and antagonist properties of RU486 and MPA in GR-mediated transrepression.

Gabapentin, an alternative drug in the prophylaxis of bipolar disorders: Preliminary data

This study describes the coupling of the recombinant rat κ-opioid receptor expressed in Chinese hamster ovary (CHO) cells to adenylyl cyclase and the effects of receptor density. The binding of [3H]diprenorphine ([3H]DPN) was dose dependent and saturable in membranes prepared from cells of early (p4–7) and late (p14–17) passage after transfection. As passage increased the receptor numbers (Bmax) declined from 231±24 (early) to 31±2 fmol/mg protein (late) but the equilibrium dissociation constant (Kd) did not change. Spiradoline dose dependently displaced [3H]DPN from membranes prepared from early and late cells revealing both high (KiH) and low (KiL) affinity binding sites. There were no significant differences in the proportion of these sites (approximately 50% KiL:50% KiH), and whilst spiradoline was generally less potent in late cells the differences were small and failed to reach statistical significance. In contrast, spiradoline produced a dose dependent inhibition of forskolin stimulated cAMP formation in whole cells with pIC50 of 8.62 and 8.00 in early compared with late cells. In addition, the maximum inhibition was dramatically reduced from 47 to 22%. Etorphine, (±)bremazocine, ICI-204,448 and (±)trans-U-50488 methanesulfonate (1 μM), compounds with activity at κ-receptors, produced a greater inhibition of cAMP formation in early (42.2, 45.8, 50.2 and 50.5%, respectively) than late (12.9, 11.8, 13.5 and 7.8%, respectively) cells, indicating that expression dependent inhibition of cAMP formation was not κ-agonist specific. Collectively, these data suggest that in CHO cells, κ-opioid receptor coupling to adenylyl cyclase is dependent on receptor expression levels.

Transformation of NIH3T3 fibroblasts by the c-Kit receptor tyrosine kinase: effect of receptor density and ligand-requirement.

Ectopic expression of the normal murine receptor tyrosine kinase, c-Kit, in NIH3T3 cells induced many phenotypic changes characteristic of transformation including anchorage-independent growth, focus formation and tumorigenicity in nude mice. Although transformation was largely dependent on the presence of recombinant murine Steel Factor (SLF), the ligand to the c-Kit receptor, anchorage independent growth did occur at a low frequency in the absence of added factor, and this could not be inhibited by neutralising antibodies or by SLF anti-sense mRNA. Clones from factor-independent colonies in semi-solid agar displayed a narrow range of c-Kit surface protein levels (4.3-6.4 x 10(4) receptors/cell) which was relatively high compared with the pool from which they were derived. Analysis of a larger series of random clones derived from adherent cultures expressing different levels of c-Kit demonstrated a positive correlation between SLF-dependent, anchorage-independent growth and c-Kit protein and mRNA expression levels (respectively, Rs = 0.58, P < 0.01; and Rs = 0.53, P < 0.01) with consistent colony formation observed with clones having > 2.5 x 10(4) receptors/cell. Interestingly, two of the three clones expressing the highest levels of c-Kit protein and mRNA produced few or no colonies in the presence or absence of SLF. Sequential overexpression of human c-KIT in NIH3T3 cells using a dihydrofolate reductase (DHFR)-encoding vector and gene co-amplification through methotrexate selection, which resulted in pools expressing up to 1.5 x 10(5) receptors/cell, confirmed that high receptor densities resulted in a decrease in colony numbers. Thus, analysis of clonal and selected populations has indicated that an optimal level of c-Kit is required for transformation of NIH3T3 cells in the presence of SLF, and that some ligand-independent transformation occurs.

Cloning and expression of human 5-HT4S receptors. Effect of receptor density on their coupling to adenylyl cyclase.

We have isolated a cDNA encoding the 5-HT4S receptor by RT-PCR on poly (A)+ RNA from both human heart and brain. The sequence homology with the rat and mouse 5-HT4 receptors was high: 93.8% of identity in the amino acid sequence. None of the 24 amino acid substitutions observed between rat and human receptors are at positions likely to modify their pharmacology. Comparing the pharmacological properties of six agonists and five antagonists on rat and human 5-HT4S receptors revealed no significant differences. We have analyzed the behavior of renzapride, a full and a partial agonist on mouse colliculi neurons and human heart biological responses respectively. The coupling efficiency of renzapride was two-fold lower than that of 5-HT for the stimulation of 5-HT4S receptors transfected in two different cell lines (LLC-PK1 and COS-7), but increasing the receptor density suppressed the partial agonist effect of renzapride.

Coupling of the cloned rat κ-opioid receptor to adenylyl cyclase is dependent on receptor expression

This study describes the coupling of the recombinant rat κ-opioid receptor expressed in Chinese hamster ovary (CHO) cells to adenylyl cyclase and the effects of receptor density. The binding of [3H]diprenorphine ([3H]DPN) was dose dependent and saturable in membranes prepared from cells of early (p4–7) and late (p14–17) passage after transfection. As passage increased the receptor numbers (Bmax) declined from 231±24 (early) to 31±2 fmol/mg protein (late) but the equilibrium dissociation constant (Kd) did not change. Spiradoline dose dependently displaced [3H]DPN from membranes prepared from early and late cells revealing both high (KiH) and low (KiL) affinity binding sites. There were no significant differences in the proportion of these sites (approximately 50% KiL:50% KiH), and whilst spiradoline was generally less potent in late cells the differences were small and failed to reach statistical significance. In contrast, spiradoline produced a dose dependent inhibition of forskolin stimulated cAMP formation in whole cells with pIC50 of 8.62 and 8.00 in early compared with late cells. In addition, the maximum inhibition was dramatically reduced from 47 to 22%. Etorphine, (±)bremazocine, ICI-204,448 and (±)trans-U-50488 methanesulfonate (1 μM), compounds with activity at κ-receptors, produced a greater inhibition of cAMP formation in early (42.2, 45.8, 50.2 and 50.5%, respectively) than late (12.9, 11.8, 13.5 and 7.8%, respectively) cells, indicating that expression dependent inhibition of cAMP formation was not κ-agonist specific. Collectively, these data suggest that in CHO cells, κ-opioid receptor coupling to adenylyl cyclase is dependent on receptor expression levels.

Effect of receptor density on the receptor-effector coupling: Use of cloned and stably expressed α1B-adrenoceptors in CHO cells

Using Chinese hamster ovary cells stably expressing α 1B-adrenoceptor as a model, we investigated whether the changes in receptor density may influence the receptor-effector coupling relationship. Among the transfected cells, two clones which showed similar pharmacological properties but markedly differed in receptor density ( B max were 1600 and 110 000 sites/cell, respectively), were examined. The phenoxybenzamine inactivation method showed that the α 1B-adrenoceptor occupancy and transients of cytosolic Ca 2+ concentration ([Ca 2+] i) response relationship was markedly nonlinear but similar in the two cell lines. The dose-response relationship for norepinephrine-induced [Ca 2+] i response showed an increase in maximum effect with no change in agonist potency, and the increase in maximum effect was disproportionate to the difference in receptor density. The results indicate that the classical model of drug-receptor action cannot appropriately describe the coupling of α 1B-adrenoceptor to [Ca 2+] i response in the single receptor expressing system.

Aggregation of IgE-receptor complexes on rat basophilic leukemia cells does not change the intrinsic affinity but can alter the kinetics of the ligand-IgE interaction.

The aggregation of IgE anchored to high-affinity Fc epsilon receptors on rat basophilic leukemia (RBL) cells by multivalent antigens initiates transmembrane signaling and ultimately cellular degranulation. Previous studies have shown that the rate of dissociation of bivalent and multivalent DNP ligands from RBL cells sensitized with anti-DNP IgE decreases with increasing ligand incubation times. One mechanism proposed for this effect is that when IgE molecules are aggregated, a conformational change occurs that results in an increase in the intrinsic affinity of IgE for antigen. This possibility was tested by measuring the equilibrium constant for the binding of monovalent DNP-lysine to anti-DNP IgE under two conditions, where the cell-bound IgE is dispersed and where it has been aggregated into visible patches on the cell surface using anti-IgE and a secondary antibody. No difference in the equilibrium constant in these two cases was observed. We also measured the rate of dissociation of a monovalent ligand from cell surface IgE under these two conditions. Whereas the affinity for monovalent ligand is not altered by IgE aggregation, we observe that the rate of ligand dissociation from IgE in clusters is slower than the rate of ligand dissociation from unaggregated IgE. These results are discussed in terms of recent theoretical developments concerning effects of receptor density on ligand binding to cell surfaces.

The effect of receptor density on the forward rate constant for binding of ligands to cell surface receptors

For monovalent ligands interacting with cell surface receptors we have directly observed the functional dependence of the forward rate constant on the number of receptors per cell (N). The experimental system we studied consisted of monovalent ligand, 2,4-dinitrophenyl (DNP)-aminocaproyl-L-tyrosine (DCT), binding to bivalent, monoclonal anti-DNP immunoglobulin E (IgE) anchored to its high affinity receptor on rat basophilic leukemia (RBL) cells. To measure the fractional occupation of antibody combining sites by DNP we employed a recently developed fluorescence technique (Erickson, J., Kane, B. Goldstein, D. Holowka, and B. Baird, 1986, Mol. Immunol., 72:769-781). Our results are well fitted by the equation (Berg and Purcell, 1977, Biophys. J., 20:193-219) konc = 4 pi DaN kappa on/[4 pi Da + N kappa on] where konc is the forward rate constant for binding to the cell, D is the diffusion constant of the ligand, a is the radius of the cell, and kappa on is the intrinsic forward rate constant describing a single IgE combining site-DNP interaction. If D is fixed at 10(-5) cm2/s, the best fit of accumulated data predicts an average cell radius of approximately 4 microns and kappa on of approximately 1.8 x 10(-13) cm3/s [1.1 x 10(8)(M . s)-1]; both in excellent agreement with RBL cell size and the single-site forward rate constant for the binding of DCT to IgE in solution, respectively. We believe this is the first report of experimental evidence that directly illustrates the effect of surface density in determining the rates of binding for small molecules to membrane receptors.

Mechanism of action of cholera toxin: effect of receptor density and multivalent binding on activation of adenylate cyclase.

Choleragen (cholera toxin) activates adenylate cyclase in HeLa cells, which contain less than 15,000 toxin receptors per cell, in a time- and concentration-dependent manner. Activation is blocked by the addition of the oligosaccharide chain of the ganglioside GM1, the receptor for the toxin. When the cells are preincubated with choleragen at 4 degrees C and then incubated with oligosaccharide at 37 degrees C, adenylate cyclase is activated less than 10%. When the preincubation phase is above 18 degrees C, adenylate cyclase becomes activated and the amount of activation depends on the time of preincubation. This inhibitory effect of the oligosaccharide is also observed with human lymphocytes and rat glial C6 cells but not with Friend erythroleukemic and mouse neuroblastoma N18 cells. The latter two cell lines have large numbers ot toxin receptors, whereas the former two cell lines have few receptors. When the number of toxin receptors in HeLa and C6 cells is increased by treating the cells with GM1, activation of adenylate cyclase by choleragen is no longer blocked by the oligosaccharide. The oligosaccharide has a corresponding effect on the displacement of bound 125I-choleragen. When bound to cells at 4 degrees C, most of the radiotoxin is displaced from HeLa, C6, and lymphocytes but not from Friend, N18, or HeLa cells pretreated with GM1. In untreated HeLa cells, dissociation of toxin-receptor complexes by the oligosaccharide depends on the time and temperature of complex formation; above 18 degrees C, the toxin rapidly becomes stably bound to the cells. The inhibitory effect of GM1 oligosaccharide us reversible, as, once it is removed, the small amount of toxin that remains bound can activate adenylate cyclase. These results are consistent with a model in which choleragen, which is multivalent, must bind to several GM1 molecules on the cell surface in order to subsequently activate adenylate cyclase. Lateral mobility of toxin-receptor complexes may be required only to achieve multivalent binding in cells with few receptors.