Ligand Depletion Research Articles

Influence of ligand binding kinetics on functional inhibition of human recombinant serotonin and norepinephrine transporters

Introduction Monoamine reuptake inhibitors treat a wide range of CNS disorders, including depression, obesity, and pain. The in vitro pharmacological properties of these inhibitors are determined routinely using radioligand binding and/or neurotransmitter uptake assays. Measurements from such studies can be influenced by assay design and ligand-specific characteristics, both of which may contribute to discrepancies in literature reports. Methods We modified traditional methodologies to identify and account for factors that can confound in vitro potency determinations. Apparent equilibrium binding affinities (p K i values) were determined in either HEK293 cells stably-transfected with human recombinant serotonin (SERT) or norepinephrine (NET) transporters, or membranes prepared from these cell lines. Care was taken to ensure that apparent affinities were measured under conditions that minimized ligand depletion and established equilibrium for both the radioligand and the compound of interest. An unlabelled ligand kinetic method was used to approximate inhibitor binding kinetic constants and corresponding dissociation half lives. To measure inhibitory effects on substrate uptake, both radiolabeled neurotransmitter ([ 3H]-5-HT or [ 3H]-NE) and fluorescence-based assays were used. The time-dependent nature of functional inhibition was examined using a fluorescent substrate uptake assay which provided real-time measurements of NET and SERT function. Results SERT and NET inhibitors displayed a range of affinities, potencies, and inhibition modes by binding and functional uptake assays. Binding kinetic profiles for this panel of inhibitors were diverse, and affected in vitro measures using the former techniques. Discussion In the present study we describe key features of in vitro assay methodology that can influence the apparent pharmacological profiles of standard SERT and/or NET inhibitors. Such information can serve as a foundation for understanding the in vitro profiles of monoamine reuptake inhibitors in the context of their clinical efficacy and tolerability.

GPS2 Is Required for Cholesterol Efflux by Triggering Histone Demethylation, LXR Recruitment, and Coregulator Assembly at the ABCG1 Locus

Transcriptional coregulators, rather than ligand signals, are suspected to confer context and pathway specificity to nuclear receptor signaling, but the identity of such specifying coregulators and the underlying molecular mechanisms remain largely enigmatic. Here we address this issue in metabolic oxysterol receptor LXR pathways and describe the selective requirement of GPS2 for ABCG1 cholesterol transporter gene transcription and cholesterol efflux from macrophages. We implicate GPS2 in facilitating LXR recruitment to an ABCG1-specific promoter/enhancer unit upon ligand activation and identify functional links to histone H3K9 demethylation. We further describe fundamental differences between ABCG1 and ABCA1 with regard to GPS2 in relation to other coregulators, which are likely to apply to additional LXR-regulated genes. Our work identifies a coregulator-dependent epigenetic mechanism governing the access of a nuclear receptor to communicating regulatory regions in the genome. The pathway and coregulator selectivity of this mechanism implies pharmacological possibilities for the development of selective LXR agonists.

Transforming Growth Factor β Depletion Is the Primary Determinant of Smad Signaling Kinetics

A cell's decision to growth arrest, apoptose, or differentiate in response to transforming growth factor beta (TGF-beta) superfamily ligands depends on the ligand concentration. How cells sense the concentration of extracellular bioavailable TGF-beta remains poorly understood. We therefore undertook a systematic quantitative analysis of how TGF-beta ligand concentration is transduced into downstream phospho-Smad2 kinetics, and we found that the rate of TGF-beta ligand depletion is the principal determinant of Smad signal duration. TGF-beta depletion is caused by two mechanisms: (i) cellular uptake of TGF-beta by a TGF-beta type II receptor-dependent mechanism and (ii) reversible binding of TGF-beta to the cell surface. Our results indicate that cells sense TGF-beta dose by depleting TGF-beta via constitutive TGF-beta type II receptor trafficking processes. Our results also have implications for the role of the TGF-beta type II receptor in disease, as tumor cells harboring TGF-beta type II receptor mutations exhibit impaired TGF-beta depletion, which may contribute to the overproduction of TGF-beta and a consequently poor prognosis in cancer.

Gd3+ binding of carboxylated polyglycidyl methacrylate latices and their colloidal stability

The binding of Gd3+ to two carboxylated polyglycidyl methacrylate latices was investigated. The latices differed in size (60 and 140 nm for CL6 and CL3, respectively) and surface charge density. The Gd3+ concentration in aqueous suspension was determined using an arsenazo (III) assay. Using 153Gd3+, the bound amount was determined directly. Because of the high binding affinity, ligand depletion became evident. The binding was pH dependent, investigated in buffer solutions not influencing the arsenazo (III) assay. Optimal binding occurs by formation of sodium salts of the carboxylic groups and replacement of Na+ and H+ by Gd3+. The dissociation constants of the particles were k D ≈ 5 × 10−5 mol/L (CL3) and 10−4 mol/L (CL6), without cooperativity (Hill plot). Colloidal stability was investigated.

Tricarbonyl Rhenium(I) Complexes Containing a Bridging 2,5-Diphenyl-1,3,4-oxadiazole Ligand: Structural, Spectroscopic, Electrochemical, and Computational Characterization

The three complexes [Re2(mu-X1)(mu-X2)(CO)6(mu-ppd-kappaN3:kappaN4)] (X1, X2 ) H, 1; X1 ) H, X2 ) Cl, 2; X1, X2 ) Cl, 3; ppd) 2,5-diphenyl-1,3,4-oxadiazole) have been synthesized by different routes, involving the reaction of [Re4(mu3-H)4(CO)12]with ppd for 1, the reaction of 1 with HCl for 2, and the reaction of [ReCl(CO)5] with ppd for 3. The three complexes possess a different number of valence electrons, so the formal Re-Re bond order varies from 2 to 1 to 0 in complexes 1, 2, and 3, respectively. This is reflected in the Re-Re bond distance (277.9, 297.9, and 358.5 pm in the same series)and in the stability of the complexes in the coordinating solvent acetonitrile (t1/2 for ppd displacement 13.6, 4.5, and 3.7 h,for 1, 2, and 3, respectively). Both experimental and calculated structures indicates that coordination induces a distortion from planarity of the diphenyloxadiazole moiety due to the interaction of the equatorial carbonyls with the bridging ppd,which increases on going from 1 to 2 to 3 (dihedral angle between the oxadiazole and the phenyl rings 18.4 degrees, 23.3 degrees, and 45.0 degrees, respectively). The UV spectra show pi-pi* transitions of the oxadiazole ligand (which shift to higher energy on increasing the distortion from the planarity, from 252 to 267 nm) and metal-to-ligand charge transfer absorptions (from 300 to 362 nm). Upon irradiation between 340 and 380 nm, complex 2 only features a weak broad emission at 527 nm(phi)0.02%), whereas upon excitation at 300 nm, the emission typical of free ppd is observed, suggesting photodissociation.Cyclic voltammetry investigations in acetonitrile showed that the three complexes exhibit ligand-centered irreversible reduction peaks (from -1.83 to -1.93 V vs Fc+|Fc), shifted to more positive values with respect to free ppd (-2.50 V). The shift however is smaller than in the analogous derivatives containing 1,2-diazines, suggesting a smaller electron depletion of the heterocycle ligand upon coordination. The complexes also show a metal-centered, bi-electronic, irreversible oxidation peak (from 1.05 to 1.37 V vs Fc+/Fc). A combined density functional and time-dependent density functional (TD DFT)study allowed us to understand the factors affecting the stability of the three complexes and to rationalize their electrochemical and photophysical properties in terms of their electronic structure.

The Impact of Orthosteric Radioligand Depletion on the Quantification of Allosteric Modulator Interactions

Radioligand binding assays remain a common method for quantifying the effects of allosteric modulators at G protein-coupled receptors. The allosteric ternary complex model (ATCM) is the simplest model applied to derive estimates of modulator affinity (K(B)) and cooperativity (alpha), which are necessary for understanding structure-activity relationships. However, the increasing drive toward assay miniaturization in modern drug discovery may lead to conditions where appreciable ligand depletion occurs in the assay. Theoretical simulations investigating the impact of orthosteric radioligand depletion on the estimation of ATCM parameters revealed the following. 1) For allosteric inhibitors, application of the standard ATCM to data obtained under depletion conditions leads to an underestimation of pK(B) and an overestimation of log alpha. 2) For allosteric enhancers, the opposite was noted, but not always; the nonlinear regression algorithm is more likely to struggle to converge to a satisfactory solution of (nondepletion) ATCM parameters in this situation. 3) Application of a novel ATCM that explicitly incorporates orthosteric ligand depletion will yield more reliable model estimates, provided the degree of depletion is not high (< approximately 50%). Subsequent experiments investigated the interaction between [3H]N-methylscopolamine and the allosteric enhancer, alcuronium, or inhibitor, gallamine, in the presence of increasing concentrations of M(2) muscarinic acetylcholine receptor and showed that application of an ATCM that explicitly incorporates radioligand depletion can indeed give more robust estimates of modulator affinity and cooperativity estimates than the standard model. These results have important implications for the quantification of allosteric modulator actions in binding-based discovery assays.

AR, the cell cycle, and prostate cancer

The androgen receptor (AR) is a critical effector of prostate cancer development and progression. The dependence of this tumor type on AR activity is exploited in treatment of disseminated prostate cancers, wherein ablation of AR function (achieved either through ligand depletion and/or the use of AR antagonists) is the first line of therapeutic intervention. These strategies are initially effective, and induce a mixed response of cell cycle arrest or apoptosis in prostate cancer cells. However, recurrent, incurable tumors ultimately arise as a result of inappropriately restored AR function. Based on these observations, it is imperative to define the mechanisms by which AR controls cancer cell proliferation. Mechanistic investigation has revealed that AR acts as a master regulator of G1-S phase progression, able to induce signals that promote G1 cyclin-dependent kinase (CDK) activity, induce phosphorylation/inactivation of the retinoblastoma tumor suppressor (RB), and thereby govern androgen-dependent proliferation. These functions appear to be independent of the recently identified TMPRSS2-ETS fusions. Once engaged, several components of the cell cycle machinery actively modulate AR activity throughout the cell cycle, thus indicating that crosstalk between the AR and cell cycle pathways likely modulate the mitogenic response to androgen. As will be discussed, discrete aberrations in this process can alter the proliferative response to androgen, and potentially subvert hormonal control of tumor progression.

Use of plasma proteins as solubilizing agents in in vitro permeability experiments: Correction for unbound drug concentration using the reciprocal permeability approach

The purpose of the present study was to explore the applicability of the reciprocal permeability approach to correct for changes in thermodynamic activity when in vitro permeability data are generated in the presence of plasma proteins. Diazepam (DIA), digoxin (DIG), and propranolol (PRO) permeability was assessed in the presence of bovine serum albumin (BSA) and bovine alpha-1-acid glycoprotein (AAG). The reciprocal permeability approach was subsequently employed to calculate the true permeability coefficient (Papp(corr)) and the operational protein association constant (nK(a)). For BSA binding, good agreement was observed between the Papp(corr) values and Papp values obtained in the absence of protein. For PRO and AAG, where binding affinity was high, deviation in the reciprocal permeability plots was evident suggesting ligand depletion at low drug/high protein concentrations. Bidirectional DIG permeability data in the presence of either BSA or AAG indicated that neither protein had an effect on the efflux transporters involved in DIG permeability. The data suggest that plasma proteins can be utilized in permeability experiments with no adverse effects on transporter function and that the reciprocal permeability approach can be used to correct permeability data for changes in unbound drug concentration.

Study of Bacteriophage T4-encoded Dam DNA (Adenine-N6)-methyltransferase Binding with Substrates by Rapid Laser UV Cross-linking

DNA methyltransferases of the Dam family (including bacteriophage T4-encoded Dam DNA (adenine-N(6))-methyltransferase (T4Dam)) catalyze methyl group transfer from S-adenosyl-L-methionine (AdoMet), producing S-adenosyl-L-homocysteine (AdoHcy) and methylated adenine residues in palindromic GATC sequences. In this study, we describe the application of direct (i.e. no exogenous cross-linking reagents) laser UV cross-linking as a universal non-perturbing approach for studying the characteristics of T4Dam binding with substrates in the equilibrium and transient modes of interaction. UV irradiation of the enzyme.substrate complexes using an Nd(3+):yttrium aluminum garnet laser at 266 nm resulted in up to 3 and >15% yields of direct T4Dam cross-linking to DNA and AdoMet, respectively. Consequently, we were able to measure equilibrium constants and dissociation rates for enzyme.substrate complexes. In particular, we demonstrate that both reaction substrates, specific DNA and AdoMet (or product AdoHcy), stabilized the ternary complex. The improved substrate affinity for the enzyme in the ternary complex significantly reduced dissociation rates (up to 2 orders of magnitude). Several of the parameters obtained (such as dissociation rate constants for the binary T4Dam.AdoMet complex and for enzyme complexes with a nonfluorescent hemimethylated DNA duplex) were previously inaccessible by other means. However, where possible, the results of laser UV cross-linking were compared with those of fluorescence analysis. Our study suggests that rapid laser UV cross-linking efficiently complements standard DNA methyltransferase-related tools and is a method of choice to probe enzyme-substrate interactions in cases in which data cannot be acquired by other means.

PET FES measures in vivo pharmacodynamics of endocrine therapy

14110 Background: Many clinical options are available for management of hormone sensitive breast cancer, including agents which lower estrogen levels such as aromatase inhibitors (AIs) and agents with block ligand binding to receptor such as tamoxifen (TAM) or fulvestrant (FUL). Estrogen receptor (ER) function is essential for sensitivity to hormonal manipulation in breast cancer treatment. We and others have previously shown that functional ER imaging using PET FES predicts response to hormonal therapy using a quantitative threshold of SUV &gt;1.5. Herein we report that FES PET provides a unique insight into in vivo pharmacodynamics of ER therapy. We hypothesized that the impact of therapy on estradiol binding to ER, measured by FES PET, differs between AIs and ER antagonists, and that early changes in receptor expression/occupancy show efficacy of drug at the tumor target. Methods: Patients undergoing treatment with AI, TAM, or FUL underwent baseline PET FDG and FES, and follow-up PET FES imaging at 2–8 weeks post initiation of therapy. Results: We observed the following changes in FES uptake on hormonal therapy: Mean percent change in FES SUV were 54% decline for TAM and FUL vs. 14% decline for AI treated patients (p&lt;.001). Patients on TAM showed complete blockade of tumor FES uptake on therapy (5/5 with residual SUV &lt;1.5), whereas patients on FUL had variable uptake and incomplete blockade at tumor sites in most patients (4/11 with residual SUV &lt;1.5) (p &lt; .05 FUL vs. TAM), despite consistent blockade of uterine FES uptake in patients where the uterus was visualized pre-FUL. Patients on AI therapy (n=14) had variable tumor uptake following treatment initiation. Conclusions: PET FES effectively monitors the in vivo activity of therapy. Estrogen blocking therapies result in a greater change in tumoral estradiol binding than in ligand depletion. TAM effectively blocks uptake of FES as would be predicted by the mechanism of action of this agent. However, FUL (while blocking uterine uptake) incompletely blocks tumor uptake, providing a mechanism to explain reduced activity of this agent in some patients. Ongoing analysis is designed to assess whether early changes in FES predict response or clinical benefit. No significant financial relationships to disclose.

Miniaturized Receptor Binding Assays: Complications Arising from Ligand Depletion

The advent of miniaturized assay formats has made possible the screening of large numbers of compounds against a single target, known as high-throughput screening. Despite this clear advantage, assay miniaturization also increases the risk of ligand depletion, where the actual concentration of free ligand is significantly lower than that added. This, in turn, complicates the interpretation of data from such assays, potentially introducing significant error if not recognized. In this study, the effects of reducing assay volume on radioligand Kd and competitor Ki values have been investigated, using the muscarinic M(3) receptor as a model system. It was found that assay miniaturization caused dramatic effects, with up to a 30-fold underestimation of ligand affinity. A theoretical model was developed and shown to accurately predict both the degree of ligand depletion in any given assay volume and the effect of this depletion on affinity estimates for competing ligands. Importantly, it was found that in most cases, errors introduced by ligand depletion could be largely corrected for by the use of appropriate analysis methods. In addition to those previously described by others, the authors propose a simple method capable of correcting errors in competition binding experiments performed in conditions of ligand depletion.

Analyzing ligand depletion in a saturation equilibrium binding experiment

I present a proposal for a laboratory practice to generate and analyze data from a saturation equilibrium binding experiment addressed to advanced undergraduate students. [(3) H]Quinuclidinyl benzilate is a nonselective muscarinic ligand with very high affinity and very low nonspecific binding to brain membranes, which contain a high density of muscarinic receptors. These features allow the instructor to devote especial emphasis to evaluate ligand depletion, and therefore, stress the subtle but fundamental difference between total (added) ligand and free ligand concentration at equilibrium.

Continuous versus intermittent tamoxifen versus intermittent/alternated tamoxifen and medroxyprogesterone acetate as first line endocrine treatment in advanced breast cancer: An EORTC phase III study (10863)

Background Continuous ligand depletion of endocrine responsive tumours may enhance resistance to therapy. Intermittent treatment with tamoxifen (T) was considered to mimic (incomplete) ligand depletion and reintroduction. Furthermore it was postulated that alternating tamoxifen with a non-cross resistant endocrine modality could (further) postpone hormone resistance. Patients and methods Postmenopausal patients with advanced breast cancer who did not progress after 4 months of first line T therapy were randomised to continue T (40 mg daily) or to 2 monthly intermittent T or intermittent/alternated T and medroxyprogesterone acetate (MPA, 300 mg daily). At progression during break or during MPA, T should be reintroduced. Endpoints of the study were progression free survival (PFS), time to resistance to tamoxifen and overall survival (OS). Results Of 593 registered patients, 276 were randomised. After 8 years follow-up the median PFS for continuous T, intermittent T and intermittent/alternated T and MPA was 11.0 (8.1–15.2), 8.0 (6.2–12.4) and 10.8 (7.1–16.7) months, respectively (NS). Resistance to tamoxifen was established only in 84%, 70% and 55% of patients in the three treatment arms, respectively. The median times from randomisation to resistance to tamoxifen were 12.5 (9.1–21.1), 13.2 (8.8–19.8) and 24.0 (16.9-60.9) months, respectively ( p < 0.001), without translation in differences in survival times. Conclusion Intermittent T or intermittent/alternated T and MPA had no impact on PFS or OS as compared with classical continuous T in patients with advanced breast cancer. Intermittent/alternated T and MPA resulted in prolonged time to resistance to T, but this might partly be due to bias by omittance of the proof of tamoxifen resistance in a high proportion of the patients in this treatment arm.

Structure and Dynamics of Membrane-associated ICP47, a Viral Inhibitor of the MHC I Antigen-processing Machinery

To evade the host's immune response, herpes simplex virus employs the immediate early gene product ICP47 (IE12) to suppress antigen presentation to cytotoxic T-lymphocytes by inhibition of the ATP-binding cassette transporter associated with antigen processing (TAP). ICP47 is a membrane-associated protein adopting an alpha-helical conformation. Its active domain was mapped to residues 3-34 and shown to encode all functional properties of the full-length protein. The active domain of ICP47 was reconstituted into oriented phospholipid bilayers and studied by proton-decoupled 15N and 2H solid-state NMR spectroscopy. In phospholipid bilayers, the protein adopts a helix-loop-helix structure, where the average tilt angle of the helices relative to the membrane surface is approximately 15 degrees (+/- 7 degrees ). The alignment of both structured domains exhibits a mosaic spread of approximately 10 degrees . A flexible dynamic loop encompassing residues 17 and 18 separates the two helices. Refinement of the experimental data indicates that helix 1 inserts more deeply into the membrane. These novel insights into the structure of ICP47 represent an important step toward a molecular understanding of the immune evasion mechanism of herpes simplex virus and are instrumental for the design of new therapeutics.

The First Structure from the SOUL/HBP Family of Heme-binding Proteins, Murine P22HBP

The First Structure from the SOUL/HBP Family of Heme-binding Proteins, Murine P22HBP

FT-IR–ATR as a tool to probe photocatalytic interfaces

The catalytic photo-oxidation on TiO 2 (Degussa P-25) of oxalic acid at pH 3.7 and of catechol at pH 6.2 has been studied by in situ ATR–FT-IR. In the case of catechol, both FT-IR and HPLC demonstrate the formation of an intermediate, the accumulation of carbonate, and the depletion of the ligand on the illuminated surface. Ligand depletion is also demonstrated by the spectral evolution of adsorbed oxalate; the data in the case also suggest that different surface complexes may inter-convert directly on the surface. These findings illustrate the potentiality of in situ ATR–FT-IR to follow the evolution of the catalytic surface. It is also clearly demonstrated that under our experimental conditions, the photolytic oxidation of the adsorbed ligands is fast as compared with the rate of exchange with the bulk. In terms of simple mechanistic considerations, the systems behave as expected for low degrees of coverage, determined by fast surface reactions, k 2{ h +} ≫ k −1, where k −1 is the rate constant for hole trapping by the adsorbed ligand, k −1 the rate constant for ligand desorption, and { h +} is the effective hole concentration, determined by the intensity of light. The rate law then turns out to be R = k 1 N S[HL], where R is the reaction rate, k 1 the adsorption rate constant, N S the surface site density, and [HL] is the bulk ligand concentration.

Ligand depletion negatively controls the mitogenic activity of epidermal growth factor

EGF activates the ErbB1 receptor, but there appears only a limited correlation between its receptor binding affinity and mitogenic activity. This is indicated by our present observation that in cells with high ErbB1 expression, including SUM102 breast tumor cells, low affinity EGF/Notch chimeras have similarly high mitogenic activity as EGF, in spite of the fact that EGF is superior in inducing receptor tyrosine phosphorylation and p42/p44 MAP-kinase activity. However, as a result of receptor-mediated internalisation high-affinity ligands such as EGF are depleted much more rapidly from the extracellular medium than low-affinity EGF/Notch chimeras. As a consequence, the mitogenic activity of EGF on ErbB1 overexpressing cells is limited by substantial degradation of internalised ligand in the period before cells enter S-phase, a phenomenon that is not observed for low affinity mutant ligands. The mitogenic activity of EGF on ErbB1 overexpressing cells does therefore not only depend on the applied concentration but also on the total amount of ligand added, and is strongly underestimated when tested in a limited assay volume. No such dependence on the incubation volume was observed for EGF activity on cells with low ErbB1 expression levels and on cells for which EGF is growth inhibitory.

Synthetic mimics of mammalian cell surface receptors: prosthetic molecules that augment living cells

Specific receptors on the surface of mammalian cells actively internalize cell-impermeable ligands by receptor-mediated endocytosis. To mimic these internalizing receptors, my laboratory is studying artificial cell surface receptors that comprise N-alkyl derivatives of 3beta-cholesterylamine linked to motifs that bind cell-impermeable ligands. When added to living mammalian cells, these synthetic receptors insert into cellular plasma membranes, project ligand-binding small molecules or peptides from the cell surface, and enable living cells to internalize targeted proteins and other cell-impermeable compounds. These artificial receptors mimic their natural counterparts by rapidly cycling between plasma membranes and intracellular endosomes, associating with proposed cholesterol and sphingolipid-rich lipid raft membrane microdomains, and delivering ligands to late endosomes/lysosomes. This "synthetic receptor targeting" strategy is briefly reviewed here and contrasted with other related cellular delivery systems. Potential applications of artificial cell surface receptors as molecular probes, agents for cellular targeting, tools for drug delivery, and methods for ligand depletion are discussed. The construction of synthetic receptors as prosthetic molecules, designed to seamlessly augment the molecular machinery of living cells, represents an exciting new frontier in the fields of bioorganic chemistry and chemical biology.

Carbon Paste Electrode Modified with Carbamoylphosphonic Acid Functionalized Mesoporous Silica: A New Mercury‐Free Sensor for Uranium Detection

AbstractThis study reports a new approach for developing a uranium electrochemical sensor that is mercury‐free, solid‐state, and has less chance for ligand depletion than existing sensors. A carbon‐paste electrode modified with carbamoylphosphonic acid self‐assembled monolayer on mesoporous silica was developed for uranium detection based on an adsorptive square‐wave stripping votammetry technique. Voltammetric responses for uranium detection are reported as a function of pH, preconcentration time, and aqueous phase uranium concentration. The uranium detection limit is 25 ppb after 5 minutes preconcentration and improved to 1 ppb after 20 minutes preconcentration. The relative standard deviations are normally less than 5%.

PH Dependence of structural stability of interleukin-2 and granulocyte colony-stimulating factor.

After a cytokine binds to its receptor on the cell surface (pH approximately 7), the complex is internalized into acidic endosomal compartments (pH approximately 5-6), where partially unfolded intermediates can form. The nature of these structural transitions was studied for wild-type interleukin-2 (IL-2) and wild-type granulocyte colony-stimulating factor (G-CSF). A noncoincidence of denaturation transitions in the secondary and tertiary structure of IL-2 and tertiary structural perturbations in G-CSF suggest the presence of an intermediate state for each, a common feature of this structural family of four-helical bundle proteins. Unexpectedly, both IL-2 and G-CSF display monotonic increases in stability as the pH is decreased from 7 to 4. We hypothesize that such cytokines with cell-based clearance mechanisms in vivo may have evolved to help stabilize endosomal complexes for sorting to lysosomal degradation. We show that mutants of both IL-2 and G-CSF have differential stabilities to their wild-type counterparts as a function of pH, and that these differences may explain the differences in ligand trafficking and depletion. Further understanding of the structural changes accompanying unfolding may help guide cytokine design with respect to ligand binding, endocytic trafficking, and, consequently, therapeutic efficacy.