Activation of nuclear receptors: a perspective from structural genomics

Crystal structures of more than two dozen different nuclear receptor ligand binding domains have defined a simple paradigm of receptor activation, in which agonist binding induces the activation function-2 (AF-2) helix to form a charge clamp for coactivator recruitment. Recent structural studies present a surprising contrast. Activation of the mouse LRH-1 receptor is independent of a bound agonist despite its large ligand binding pocket, whereas the activation of the Drosophila DHR38 receptor is dependent on ecdysteroids even though the receptor lacks a ligand binding pocket. These new findings shed light on the diverse structural mechanisms that nuclear receptors have evolved for activation, and have important implications in their respective signaling pathways.     

CR1/CR2 interactions modulate the functions of the cell surface epidermal growth factor receptor

Recent crystallographic data on the isolated extracellular domain of the epidermal growth factor receptor (EGFR) have suggested a model for its activation by ligand. We have tested this model in the context of the full-length EGFR displayed at the cell surface, by introducing mutations in two regions (CR1 and CR2) of the extracellular domain thought to be critical for regulation of receptor activation. Mutations in the CR1 and CR2 domains have opposing effects on ligand binding affinity, receptor dimerization, tyrosine kinase activation, and signaling competence. Tyr(246) is a critical residue in the CR1 loop, which is implicated in the positioning and stabilization of the receptor dimer interface after ligand binding; mutations of Tyr(246) impair or abolish receptor function. Mutations in CR2, which weaken the interaction that restricts the receptor to the tethered (inactive) state, enhance responsiveness to EGF by increasing affinity for the ligand. However, weakening of the CR1/CR2 interaction does not result in spontaneous activation of the receptors' kinase. We have used an antibody (mAb 806), which recognizes a transition state of the EGF receptor between the negatively constrained, tethered state and the fully active back-to-back dimer conformation, to follow conformational changes in the wild-type and mutant EGF receptors after ligand binding. Our results suggest that EGFR on the cell surface can be untethered, but this form is inactive; thus, untethering of the receptor is not sufficient for activation, and ligand binding is essential for the correct positioning of the two receptor subunits to achieve kinase activation.     

Understanding the mechanism of insulin and insulin-like growth factor (IGF) receptor activation by IGF-II

Background

Insulin-like growth factor-II (IGF-II) promotes cell proliferation and survival and plays an important role in normal fetal development and placental function. IGF-II binds both the insulin-like growth factor receptor (IGF-1R) and insulin receptor isoform A (IR-A) with high affinity. Interestingly both IGF-II and the IR-A are often upregulated in cancer and IGF-II acts via both receptors to promote cancer proliferation. There is relatively little known about the mechanism of ligand induced activation of the insulin (IR) and IGF-1R. The recently solved IR structure reveals a folded over dimer with two potential ligand binding pockets arising from residues on each receptor half. Site-directed mutagenesis has mapped receptor residues important for ligand binding to two separate sites within the ligand binding pocket and we have recently shown that the IGFs have two separate binding surfaces which interact with the receptor sites 1 and 2.

Methodology/Principal Findings

In this study we describe a series of partial IGF-1R and IR agonists generated by mutating Glu12 of IGF-II. By comparing receptor binding affinities, abilities to induce negative cooperativity and potencies in receptor activation, we provide evidence that residue Glu12 bridges the two receptor halves leading to receptor activation.

Conclusions/Significance

This study provides novel insight into the mechanism of receptor binding and activation by IGF-II, which may be important for the future development of inhibitors of its action for the treatment of cancer.     

Mechanisms for activation and antagonism of an AMPA-sensitive glutamate receptor: crystal structures of the GluR2 ligand binding core

Crystal structures of the GluR2 ligand binding core (S1S2) have been determined in the apo state and in the presence of the antagonist DNQX, the partial agonist kainate, and the full agonists AMPA and glutamate. The domains of the S1S2 ligand binding core are expanded in the apo state and contract upon ligand binding with the extent of domain separation decreasing in the order of apo > DNQX > kainate > glutamate approximately equal to AMPA. These results suggest that agonist-induced domain closure gates the transmembrane channel and the extent of receptor activation depends upon the degree of domain closure. AMPA and glutamate also promote a 180 degrees flip of a trans peptide bond in the ligand binding site. The crystal packing of the ligand binding cores suggests modes for subunit-subunit contact in the intact receptor and mechanisms by which allosteric effectors modulate receptor activity.     

Lipid-dependent sequential allosteric activation of heat-sensing TRPV1 channels by anchor-stereoselective “hot” vanilloid compounds and analogs

Both a silent resident phosphatidylinositol lipid and a “hot” vanilloid agonist capsaicin or resiniferatoxin have been shown to share the same inter-subunit binding pocket between a voltage sensor like domain and a pore domain in TRPV1. However, how the vanilloid competes off the resident lipid for allosteric TRPV1 activation is unknown. Here, the in silico research suggested that anchor-stereoselective sequential cooperativity between an initial recessive transient silent weak ligand binding site and a subsequent dominant steady-state strong ligand binding site in the vanilloid pocket may facilitate the lipid release for allosteric activation of TRPV1 by vanilloids or analogs upon non-covalent interactions. Thus, the resident lipid may play a critical role in allosteric activation of TRPV1 by vanilloid compounds and analogs.     

Structural and energetic analysis of activation by a cyclic nucleotide binding domain

MlotiK1 is a prokaryotic homolog of cyclic-nucleotide-dependent ion channels that contains an intracellular C-terminal cyclic nucleotide binding (CNB) domain. X-ray structures of the CNB domain have been solved in the absence of ligand and bound to cAMP. Both the full-length channel and CNB domain fragment are easily expressed and purified, making MlotiK1 a useful model system for dissecting activation by ligand binding. We have used X-ray crystallography to determine three new MlotiK1 CNB domain structures: a second apo configuration, a cGMP-bound structure, and a second cAMP-bound structure. In combination, the five MlotiK1 CNB domain structures provide a unique opportunity for analyzing, within a single protein, the structural differences between the apo state and the bound state, and the structural variability within each state. With this analysis as a guide, we have probed the nucleotide selectivity and importance of specific residue side chains in ligand binding and channel activation. These data help to identify ligand-protein interactions that are important for ligand dependence in MlotiK1 and, more globally, in the class of nucleotide-dependent proteins.     

Evidence that activation of platelet calpain is induced as a consequence of binding of adhesive ligand to the integrin, glycoprotein IIb-IIIa

Calpain (a Ca(2+)-dependent protease) is present in many cell types. Because it is present in the cytosol, the potential exists that it may regulate critical intracellular events by inducing crucial proteolytic cleavages. However, the concentrations of Ca2+ required to activate calpain are higher than those attained in the cytoplasm of most cells. Thus, the physiological importance of calpain and the mechanisms involved in its activation have remained elusive. In this study, we show that calpain rapidly moved to a peripheral location upon the addition of an agonist to suspensions of platelets, but it remained unactivated. We provide three lines of evidence that calpain was subsequently activated by a mechanism that required the binding of an adhesive ligand to the major platelet integrin, glycoprotein (GP) IIb- IIIa: calpain activation was prevented by RGDS, a tetrapeptide that inhibits the binding of adhesive ligand to GP IIb-IIIa; it was also prevented by monoclonal antibodies that inhibit adhesive ligand binding to GP IIb-IIIa; and its activation was markedly reduced in platelets from patients whose platelets have greatly reduced levels of functional GP IIb-IIIa. Thus, in platelets, binding of the extracellular domain of GP IIb-IIIa to its adhesive ligand can initiate a transmembrane signal that activates intracellular calpain. Because calpain is present in focal contacts of adherent cells, the interaction of integrins with adhesive ligands in the extracellular matrix may regulate activation of calpain in other cell types as well.     

Ligand displaces heat shock protein 90 from overlapping binding sites within the aryl hydrocarbon receptor ligand-binding domain

Hsp90 (heat shock protein of 90 kDa) is often found associated with functional domains of client proteins, including those for ligand binding, dimerization, DNA binding, and enzymatic activity. Although Hsp90 can maintain the conformation of functionally important domains prior to activation of the client protein, its specific binding site and the mechanism(s) of Hsp90 dissociation during activation are unknown. Here, we have identified and characterized residues involved in Hsp90 binding within the aryl hydrocarbon receptor (AhR) ligand-binding domain and demonstrate that they overlap with those involved in ligand binding. In agreement with this spatial model, ligand binding results in Hsp90 dissociation from the AhR Per-ARNT-Sim B fragment. Interestingly, whereas Hsp90-binding residues within the ligand-binding domain were not involved in Hsp90-dependent AhR protein stability, several of these residues are important for ligand-dependent AhR activation, and their mutation resulted in conversion of two AhR antagonists/partial agonists into full AhR agonists. These studies reveal co-localization of a tentative Hsp90-binding site with that for AhR ligand binding and provide the first molecular mechanism for Hsp90 dissociation in the activation of a client protein.     

Activation of transmembrane cell‐surface receptors via a common mechanism? The “rotation model”

It has long been thought that transmembrane cell‐surface receptors, such as receptor tyrosine kinases and cytokine receptors, among others, are activated by ligand binding through ligand‐induced dimerization of the receptors. However, there is growing evidence that prior to ligand binding, various transmembrane receptors have a preformed, yet inactive, dimeric structure on the cell surface. Various studies also demonstrate that during transmembrane signaling, ligand binding to the extracellular domain of receptor dimers induces a rotation of transmembrane domains, followed by rearrangement and/or activation of intracellular domains. The paper here describes transmembrane cell‐surface receptors that are known or proposed to exist in dimeric form prior to ligand binding, and discusses how these preformed dimers are activated by ligand binding.     

Comparison of ligand polarization and enzyme activation in medium- and short-chain acyl-coenzyme A dehydrogenase-novel analog complexes

Spectroelectrochemical and off-resonance Raman indicate that substrate/product binding to medium-chain acyl-coenzyme A (CoA) dehydrogenase (pMCAD) results in ligand polarization and positive flavin potential shifts, which activate the enzyme for electron transfer. Bacterial short-chain acyl-CoA dehydrogenase (bSCAD) typically exhibits smaller potential shifts upon substrate/product binding that have not been linked to ligand polarization. To further investigate the roles of ligand binding and polarization in activation, several novel aromatic carboxyloyl-CoAs were designed. These analogs allowed for the first direct comparison of pMCAD and bSCAD mechanisms. The results indicate that pMCAD activation can occur without perceptible analog polarization. bSCAD data provide the first spectral evidence of ligand polarization. The potential alterations exhibited by ligand-bound bSCAD are smaller than those of pMCAD, while their directionality and magnitude suggest differing enzyme-analog interactions. Such data provide the first indication of variations in the activation mechanism of these enzymes, which were thought to be comparable in both structure and function.     

Soluble Interleukin-5 Receptor α-Chain Binding Assays: Use for Screening and Analysis of Interleukin-5 Mutants

Interleukin-5 (IL-5) is a key cytokine for the production, differentiation, and activation of eosinophils. IL-5 is a member of the four helical bundle family of cytokines, and in common with many members of the cytokine family it binds to a heterodimeric receptor composed of a ligand binding α-chain and a signal-transducing β-chain. We have established two receptor/ligand binding assays based on the extracellular domain of the receptor α-chain which we have produced as a fusion protein. One assay is based on scintillation proximity fluoromicrospheres and radiolabeled ligand and the other on detection of biotinylated ligand binding to immobilized receptor using a chemiluminescent substrate in a 96-well microtiter plate format. Both receptor binding assays have been optimized for high throughput screening for receptor antagonists. These assays were also used for analytical purposes and the binding of ligand to the receptor α-chain was compared directly to receptor binding assays performed on TF-1 cells which express the receptor αβ-heterodimer. These three assays have been used to study site-directed mutants of IL-5 to determine the important residues for interaction of the cytokine with each chain of the receptor (P. Graber et al. (1995) J. Biol. Chem. 270, 15762-15769).     

Predictive features of ligand‐specific signaling through the estrogen receptor

Some estrogen receptor‐α (ERα)‐targeted breast cancer therapies such as tamoxifen have tissue‐selective or cell‐specific activities, while others have similar activities in different cell types. To identify biophysical determinants of cell‐specific signaling and breast cancer cell proliferation, we synthesized 241 ERα ligands based on 19 chemical scaffolds, and compared ligand response using quantitative bioassays for canonical ERα activities and X‐ray crystallography. Ligands that regulate the dynamics and stability of the coactivator‐binding site in the C‐terminal ligand‐binding domain, called activation function‐2 (AF‐2), showed similar activity profiles in different cell types. Such ligands induced breast cancer cell proliferation in a manner that was predicted by the canonical recruitment of the coactivators NCOA1/2/3 and induction of the GREB1 proliferative gene. For some ligand series, a single inter‐atomic distance in the ligand‐binding domain predicted their proliferative effects. In contrast, the N‐terminal coactivator‐binding site, activation function‐1 (AF‐1), determined cell‐specific signaling induced by ligands that used alternate mechanisms to control cell proliferation. Thus, incorporating systems structural analyses with quantitative chemical biology reveals how ligands can achieve distinct allosteric signaling outcomes through ERα.     

Structural activation pathways from dynamic olfactory receptor-odorant interactions

We have simulated an odor ligand's dynamic behavior in the binding region of an olfactory receptor (OR). Our short timescale computational studies (up to 200 ps) have helped identify unprecedented postdocking ligand behavior of ligands. From in vacuo molecular dynamics simulations of interactions between models of rat OR I7 and 10 aldehyde ligands, we have identified a dissociative pathway along which the ligand exits and enters the OR-binding pocket--a transit event. The ligand's transit through the receptor's binding region may mark the beginning of a signal transduction cascade leading to odor recognition. We have graphically traced the rotameric changes in key OR amino acid side chains during the transit. Our results have helped substantiate or refute previously held notions of amino acid contribution to ligand stability in the binding pocket. Our observations of ligand activity when compared to those of experimental (electroolfactogram response) OR-activation studies provide a view to predicting the stability of ligands in the binding pocket as a precursor to OR activation by the ligand.     

Signaling pathways of transforming growth factor beta family members

Transforming growth factor beta (TGF-beta) signaling controls varies of cellular processes, including cell proliferation, differentiation, fibrosis, apoptosis and specification of developmental fate during embryogenesis as well as in mature tissues. The members of TGF-betas family are secreted as inactive (latent) precursors, what prevents uncontrolled activation of the cognate receptors. After activation TGF-beta ligand initiates signaling by binding to and bringing together type I and type II receptor serine/threronine kinases on the cell surface. Recent cellular, biochemical and structural studies have revealed significant insight into the mechanisms of the activation of TGF-beta receptors through ligand binding, the activation of Smad proteins through phosphorylation as well as Smad independent pathways.     

cAMP-induced desensitization of surface cAMP receptors in Dictyostelium: different second messengers mediate receptor phosphorylation, loss of ligand binding, degradation of receptor, and reduction of receptor mRNA levels.

Surface cAMP receptors on Dictyostelium cells are linked to several second messenger systems and mediate multiple physiological responses, including chemotaxis and differentiation. Activation of the receptor also triggers events which desensitize signal transduction. These events include the following: 1) loss of ligand binding without loss of receptor protein; 2) phosphorylation of the receptor protein, which may lead to impaired signal transduction; 3) redistribution and degradation of the receptor protein; and 4) decrease of cyclic AMP (cAMP) receptor mRNA levels. These mechanisms of desensitization were investigated with the use of mutant synag7, with no activation of adenylyl cyclase; fgdC, with no activation of phospholipase C; and fgdA, with defects in both pathways. cAMP-induced receptor phosphorylation and loss of ligand binding activity was normal in all mutants. In contrast, cAMP-induced degradation of the receptor was absent in all mutants. The cAMP-induced decrease of cAMP-receptor mRNA levels was normal in mutant synag7, but absent in mutant fgdC. Finally, the cAMP analogue (Rp)-cAMPS induced loss of ligand binding without inducing second messenger responses or phosphorylation, redistribution, and degradation of the receptor. We conclude that 1) loss of ligand binding can occur in the absence of receptor phosphorylation; 2) loss of ligand binding and receptor phosphorylation do not require the activation of second messenger systems; 3) cAMP-induced degradation of the receptor may require the phosphorylation of the receptor as well as the activation of at least the synag7 and fgdC gene products; and 4) cAMP-induced decrease of receptor mRNA levels requires the activation of the fgdC gene product and not the synag7 gene product. These results imply that desensitization is composed of multiple components that are regulated by different but partly overlapping sensory transduction pathways.     

Kinetic models for association of 2,3,7,8-tetrachlorodibenzo-p-dioxin with the Ah receptor

Saturation binding studies of the interaction between 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the Ah receptor obtained from the hepatic cytosol of Wistar rats have been carried out. The conventional Scatchard analysis for determination of the equilibrium constant for ligand-receptor binding has been shown to be inappropriate due to thermal inactivation of the unoccupied receptor. Simulation models of the receptor-ligand binding kinetics which take into account receptor degradation have been developed and the results are consistent with two alternative kinetic models. In Model 1, reversible 2,3,7,8-TCDD-receptor binding occurs in parallel with inactivation of the unbound receptor; analysis of the observed data using this model suggests that the previously determined equilibrium constants (Kass) for association of the ligand with the receptor are orders of magnitude too low and the total initial receptor concentrations are somewhat underestimated. In Model 2, the unbound receptor is converted unimolecularly to an activated state which then undergoes competitive degradation or entrapment by ligand. Experiments have been carried out over the temperature range 4-37 degrees C, enabling activation parameters to be obtained. According to Scheme 1, the activation enthalpies for association of receptor with ligand and for thermal inactivation of the unoccupied receptor are high, and numerically almost identical (delta H++ ca 125 kJ mol-1). These reactions are strongly entropically driven and this is consistent with association being accompanied by a conformational change in the receptor protein, and the previously postulated binding of the ligand to a hydrophobic pocket. According to Scheme 2, there is only one enthalpy of activation because both inactivation and entrapment by 2,3,7,8-TCDD are fast processes which follow the same slow activation step. On the basis of this latter model, a 10(-9) M concentration of 2,3,7,8-TCDD is sufficient to trap roughly two-thirds of the activated receptors.     

Is the isolated ligand binding domain a good model of the domain in the native receptor?

Numerous studies have used the atomic level structure of the isolated ligand binding domain of the glutamate receptor to elucidate the agonist-induced activation and desensitization processes in this group of proteins. However, no study has demonstrated the structural equivalence of the isolated ligand binding fragments and the protein in the native receptor. In this report, using visible absorption spectroscopy we show that the electronic environment of the antagonist 6-cyano-7-nitro-2,3-dihydroxyquinoxaline is identical for the isolated protein and the native glutamate receptors expressed in cells. Our results hence establish that the local structure of the ligand binding site is the same in the two proteins and validate the detailed structure-function relationships that have been developed based on a comparison of the structure of the isolated ligand binding domain and electrophysiological consequences in the native receptor.     

ATP modulation of the ligand binding and signal transduction activities of the type C natriuretic peptide receptor guanylate cyclase

The type C natriuretic peptide (CNP)-activated guanylate cyclase (CNP-RGC) is a single-chain transmembrane-spanning protein, containing both CNP binding and catalytic cyclase activities. Upon binding CNP to the extracellular receptor domain, the cytosolic catalytic domain of CNP-RGC is activated, generating the second messenger cyclic GMP. Obligatory in this activation process is an intervening signal transduction step which is regulated by ATP binding to the cyclase. This bridges the events of ligand binding and cyclase activation. A defined sequence motif (Gly⁴⁹⁹-Xa-Xa-Xa-Gly⁵⁰³), termed ATP regulatory module (ARM), is critical for this step. The present study shows that ATP not only amplifies the signal transduction step, it also concomitantly reduces the ligand binding activity of CNP-RGC. Reduction in the ligand binding activity is a consequence of the transformation of the high affinity receptor-form to the low affinity receptor-form. A single ARM residue Gly⁴⁹⁹ is critical in the mediation of both ATP effects, signal transduction and ligand binding activity of the receptor. Thus, this residue represents an ATP bimodal switch to turn the CNP signal on and off.