Circular permutation of the granulocyte colony-stimulating factor receptor agonist domain of myelopoietin

Myelopoietins (MPOs) are a family of engineered dual interleukin-3 (IL-3) and granulocyte colony-stimulating factor (G-CSF) receptor agonists that are superior in comparison to the single agonists in their ability to promote the growth and maturation of hematopoietic cells of the myeloid lineage. A series of MPO molecules were created which incorporated circularly permuted G-CSF (cpG-CSF) sequences with an IL-3 receptor (IL-3R) agonist moiety attached at locations that correspond to the loops that connect the helices of the G-CSF four-helix bundle structure. The cpG-CSF linkage sites (using the original sequence numbering) were residue 39, which is at the beginning of the first loop connecting helices 1 and 2; residue 97, which is in the turn connecting helices 2 and 3; and residues 126, 133, and 142, which are at the beginning, middle, and end, respectively, of the loop connecting helices 3 and 4. The N- and C-terminal helices of each cpG-CSF domain were constrained, either by direct linkage of the termini (L0) or by replacement of the amino-terminal 10-residue segment with a seven-residue linker composed of SGGSGGS (L1). All of the MPO molecules stimulated the proliferation of both IL-3-dependent (EC50 = 13-95 pM) and G-CSF-dependent (EC50 = 35-710 pM) cell lines. MPOs with the IL-3R agonist domain linked to cpG-CSFs in the first (residue 39) or second (residue 133) long overhand loops were found by CD spectroscopy to have helical contents similar to that expected for a protein comprised of two linked four-helix bundles. The MPOs retained the ability to bind to the IL-3R with affinities similar to that of the parental MPO. Using both a cell surface competitive binding assay and surface plasmon resonance detection of binding kinetics, the MPOs were found to bind to the G-CSF receptor with low nanomolar affinities, similar to that of G-CSF(S17). In a study of isolated cpG-CSF domains [Feng, Y., et al. (1999) Biochemistry 38, 4553-4563], domains with the L1 linker had lower G-CSF receptor-mediated proliferative activities and conformational stabilities than those which had the L0 linker. A similar trend was found for the MPOs in which the G-CSFR agonist activity is mostly a property of the cpG-CSF domain. Important exceptions were found in which the linkage to the IL-3R agonist domain either restored (e.g., attachment at residue 142) or further decreased (linkage at residue 39) the G-CSFR-mediated proliferative activity. MPO in which the IL-3R agonist domain is attached to the cpG-CSF(L1)[133/132] domain was shown to be more potent than the coaddition of the IL-3R agonist and G-CSF in stimulating the production of CFU-GM colonies in a human bone marrow-derived CD34+ colony-forming unit assay. Several MPOs also had decreased proinflammatory activity in a leukotriene C4 release assay using N-formyl-Met-Leu-Phe-primed human monocytes. It was found that circular permutation of the G-CSF domain can alter the ratio of G-CSFR:IL-3R agonist activities, demonstrating that it is a useful tool in engineering chimeric proteins with therapeutic potential.     

Microscopic kinetics and energetics distinguish GABA(A) receptor agonists from antagonists

Although agonists and competitive antagonists presumably occupy overlapping binding sites on ligand-gated channels, these interactions cannot be identical because agonists cause channel opening whereas antagonists do not. One explanation is that only agonist binding performs enough work on the receptor to cause the conformational changes that lead to gating. This idea is supported by agonist binding rates at GABA(A) and nicotinic acetylcholine receptors that are slower than expected for a diffusion-limited process, suggesting that agonist binding involves an energy-requiring event. This hypothesis predicts that competitive antagonist binding should require less activation energy than agonist binding. To test this idea, we developed a novel deconvolution-based method to compare binding and unbinding kinetics of GABA(A) receptor agonists and antagonists in outside-out patches from rat hippocampal neurons. Agonist and antagonist unbinding rates were steeply correlated with affinity. Unlike the agonists, three of the four antagonists tested had binding rates that were fast, independent of affinity, and could be accounted for by diffusion- and dehydration-limited processes. In contrast, agonist binding involved additional energy-requiring steps, consistent with the idea that channel gating is initiated by agonist-triggered movements within the ligand binding site. Antagonist binding does not appear to produce such movements, and may in fact prevent them.     

Effect of novel N-aryl sulfonamide substituted 3-morpholino arecoline derivatives as muscarinic receptor 1 agonists in Alzheimer's dementia models

A series of novel, potent, and selective muscarinic receptor 1 agonists (M1 receptor agonists) that employ a key N-substituted morpholine Arecoline moiety has been synthesized as part of research effort for the therapy of Alzheimer’s diseases. The ester group of arecoline (which is reported as muscarinic agonist) has been replaced by N-substituted morpholine ring. The structure–activity relationship reveals that the electron donating 4-substituted sulfonyl derivatives (9a, 9b, 9c, and 9e) on the nitrogen atom of the morpholine ring increases the affinity of M1 receptor binding 50- to 80-fold greater than the corresponding arecoline. Other derivatives also showed considerable M1 receptor binding affinity.     

Prostanoid receptor expression by human airway smooth muscle cells and regulation of the secretion of granulocyte colony-stimulating factor

The prostanoid receptors on human airway smooth muscle cells (HASMC) that augment the release by IL-1beta of granulocyte colony-stimulating factor (G-CSF) have been characterized and the signaling pathway elucidated. PCR of HASM cDNA identified products corresponding to EP(2), EP(3), and EP(4) receptor subtypes. These findings were corroborated at the protein level by immunocytochemistry. IL-1beta promoted the elaboration of G-CSF, which was augmented by PGE(2). Cicaprost (IP receptor agonist) was approximately equiactive with PGE(2), whereas PGD(2), PGF(2alpha), and U-46619 (TP receptor agonist) were over 10-fold less potent. Neither SQ 29,548 nor BW A868C (TP and DP(1) receptor antagonists, respectively) attenuated the enhancement of G-CSF release evoking any of the prostanoids studied. With respect to PGE(2), the EP receptor agonists 16,16-dimethyl PGE(2) (nonselective), misoprostol (EP(2)/EP(3) selective), 17-phenyl-omega-trinor PGE(2) (EP(1) selective), ONO-AE1-259, and butaprost (both EP(2) selective) were full agonists at enhancing G-CSF release. AH 6809 (10 microM) and L-161,982 (2 microM), which can be used in HASMC as selective EP(2) and EP(4) receptor antagonists, respectively, failed to displace to the right the PGE(2) concentration-response curve that described the augmented G-CSF release. In contrast, AH 6809 and L-161,982 in combination competitively antagonized PGE(2)-induced G-CSF release. Augmentation of G-CSF release by PGE(2) was mimicked by 8-BrcAMP and abolished in cells infected with an adenovirus vector encoding an inhibitor protein of cAMP-dependent protein kinase (PKA). These data demonstrate that PGE(2) facilitates G-CSF secretion from HASMC through a PKA-dependent mechanism by acting through EP(2) and EP(4) prostanoid receptors and that effective antagonism is realized only when both subtypes are blocked concurrently.     

Resiniferatoxin binds to the capsaicin receptor (TRPV1) near the extracellular side of the S4 transmembrane domain

The capsaicin receptor (TRPV1) is a nonselective cation channel that is activated in nociceptors by several painful stimuli, and hence TRPV1 antagonists could represent a novel class of analgesic compounds. Resiniferatoxin (RTX), a potent agonist of TRPV1, and iodoresiniferatoxin (I-RTX), a potent antagonist of TRPV1, both bind with higher affinity to the rat TRPV1 (rTRPV1) than the human (hTRPV1) isoform. To identify the structural features responsible for this difference in affinity, [(3)H]RTX binding to chimeras between hTRPV1 and rTRPV1 was characterized. The "sensor" region within the transmembrane domain (S1-S4) was found to determine [(3)H]RTX binding affinity. All 16 different residues in this region were systematically substituted in hTRPV1 with rTRPV1 residues. A single mutation in the S4 membrane domain of hTRPV1, L547M, caused a 30-fold increase in [(3)H]RTX affinity whereas the inverse mutation in rTRPV1, M547L, caused a 30-fold decrease in affinity for [(3)H]RTX, and several other agonists and antagonists were similarly affected by these mutations. TRPV1 channels with mutations at position 547 were expressed in oocytes, and the relative response to RTX followed a pattern similar to that seen with [(3)H]RTX binding. These data suggest a model where Met-547 in the S4 domain of TRPV1 forms a binding pocket with Tyr-511 in the S3 domain. This model places RTX near the sensor domain thought to move during the gating process and should help to guide further work designed to understand the gating mechanisms of TRPV1 channels based on comparisons between the agonist RTX and the related competitive antagonist I-RTX.     

Agonist-induced isomerization of the alpha 1-adrenergic receptor: kinetic analysis using broken-cell and solubilized preparations

The affinity of agonists but not antagonists at hepatic membrane alpha 1-adrenergic receptors is temperature dependent; a 100-fold higher affinity is observed at 4 degrees C than at 37 degrees C. The relationship between these two agonist affinity states was investigated by using a strategy that allows the kinetics of this transition to be examined under equilibrium conditions. When competition assays are performed at 37 degrees C for varying intervals and the reaction mixture is then rapidly cooled by freezing, allowed to thaw, and further equilibrated at 4 degrees C, a rapid and progressive decrease (t1/2 of 1-2 min) in agonist affinity occurs, the extent of which is directly related to the incubation time at 37 degrees C. This decrease in agonist affinity is sustained as long as agonist is present but can be reversed by its subsequent removal. In contrast, no change in affinity is seen in identical experiments when antagonists are employed as the competing ligand. High-affinity binding of agonists is also demonstrated in short-term nonequilibrium experiments, indicating that the low-temperature incubations do not induce, but rather stabilize, a receptor conformation of high affinity for agonists. These findings suggest that the predominantly low-affinity binding of agonists to alpha 1-adrenergic receptors demonstrated in equilibrium studies at physiological temperatures may be the result of a ligand-driven decrease in affinity. Since the transition in receptor affinity for agonists occurs not only in broken-cell preparations but also after detergent solubilization of the membrane receptor, it most likely is due to an agonist-induced change in the conformation of the receptor protein per se.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutation W284L of the human delta opioid receptor reveals agonist specific receptor conformations for G protein activation

Intrinsic activities of different delta opioid agonists were determined in a [35S]GTPgammaS binding assay using cell membranes from Chinese hamster ovary (CHO) cells stably expressing the wild type (hDOR/CHO) or W284L mutant human delta opioid receptor (W284L/CHO). Agonist binding affinities were regulated more robustly by sodium and guanine nucleotide in W284L/CHO than in hDOR/ CHO cell membranes. The W284L mutation selectively reduced the affinity of SNC 80 while having moderate effect ((-) TAN 67) or no effect (DPDPE) on the affinities of other delta selective agonists. The mutation had opposite effects on the intrinsic activities of agonists belonging to different chemical classes. The effects of the mutation on agonist affinities and potencies were independent from its effects on the intrinsic activities of the agonists. Maximal stimulation of [35S]GTPgammaS binding by SNC 80 was 2-fold higher in W284L mutant cell membranes than in wild type hDOR/CHO cell membranes, despite lower receptor expression levels in the W284L/CHO cells. The binding affinity of SNC 80 however, was significantly reduced (15-fold and 30-fold in the absence or presence of sodium+GDP respectively) in W284L/CHO cell membranes relative to wild type hDOR/CHO membranes. Conversely, the Emax of (-)TAN 67 in the [35S]GTPgammaS binding assay was markedly reduced (0.6-fold of that of the wild type) with only a slight (6-fold) reduction in its binding affinity. The affinity and intrinsic activity of DPDPE on the other hand remained unchanged at the W284L mutant hDOR. The mutation had similar effects on the affinities potencies and intrinsic activities of (-)TAN 67 and SB 219825. The results indicate that delta opioid agonists of different chemical classes use specific conformations for G protein activation.     

Anterior pituitary dopamine receptors. Demonstration of interconvertible high and low affinity states of the D-2 dopamine receptor

The interactions of dopaminergic agonists and antagonists with binding sites in bovine anterior pituitary membranes have been investigated with radioligand-binding techniques and computer-modeling procedures. 3H-labeled agonist binding is stereospecific, reversible, saturable, and of high affinity. The rank order of catecholamines, phenothiazines, and related drugs in competing for 3H-agonist binding is indicative of interactions with a D-2 dopamine receptor. Both agonist/3H-agonist and antagonist/3H-agonist competition curves are monophasic and noncooperative (nH = 1) with computer analysis indicating a single class of binding sites. Specific 3H-agonist binding can be completely inhibited by guanine nucleotides. GppNHp us the most potent nucleotide followed by GTP and GDP which are equipotent. The equilibrium binding capacity for 3H-labeled antagonists is twice that for 3H-agonists. Unlabeled antagonists inhibit 3H-antagonist binding competitively and exhibit antagonist/3H-antagonist competition curves which model best to a state of homogeneous affinity. In contrast, unlabeled agonists inhibit 3H-antagonist binding in a heterogeneous fashion displaying multiphasic (nH less than 1) competition curves which can be resolved into high and low affinity binding sites. In the presence of saturating concentrations of guanine nucleotides, however, the agonist/3H-antagonist curves model best to a single affinity state which is identical with the low affinity state seen in control curves. The binding data can be explained by postulating two states of the D-2 dopamine receptor, inducible by agonists but not antagonists and modulated by guanine nucleotides.     

Truncation of the extended carboxyl-terminal domain increases the expression and regulatory activity of the avian beta-adrenergic receptor.

A series of mutant avian beta-adrenergic receptors with progressively truncated carboxyl termini have been expressed in insect and mammalian cells. Removal of 18-124 amino acid residues caused multiple phenotypic changes in the receptor. Membranes from cells that expressed the truncated receptors displayed elevated basal (2- to 3-fold) and agonist-stimulated adenylylcyclase activities. Adenylylcyclase activity in these membranes also displayed greater stimulation in response to partial agonists. Activity was also markedly stimulated by beta-adrenergic ligands that are usually considered to be antagonists (alprenolol, greater than 4-fold; propranolol, approximately 2-fold). Wild type receptor did not mediate a response to these classical antagonists. After purification and reconstitution with Gs, the truncated receptors did not appear to be more active than the wild type. Guanine nucleotides modulated the affinity of agonist for the truncated receptors, whereas the affinity of agonist for the wild type receptor was not altered by guanine nucleotides. The truncated receptors were solubilized from the membrane more efficiently and were more susceptible to amino-terminal proteolysis than was the wild type protein. These results suggest interaction of the carboxyl terminus of the avian beta-adrenergic receptor with cellular regulatory or structural elements.     

Characterization of a novel, non-peptidyl antagonist of the human glucagon receptor

We have identified a series of potent, orally bioavailable, non-peptidyl, triarylimidazole and triarylpyrrole glucagon receptor antagonists. 2-(4-Pyridyl)-5-(4-chlorophenyl)-3-(5-bromo-2-propyloxyphenyl)p yrr ole (L-168,049), a prototypical member of this series, inhibits binding of labeled glucagon to the human glucagon receptor with an IC50 = 3. 7 +/- 3.4 nM (n = 7) but does not inhibit binding of labeled glucagon-like peptide to the highly homologous human glucagon-like peptide receptor at concentrations up to 10 microM. The binding affinity of L-168,049 for the human glucagon receptor is decreased 24-fold by the inclusion of divalent cations (5 mM). L-168,049 increases the apparent EC50 for glucagon stimulation of adenylyl cyclase in Chinese hamster ovary cells expressing the human glucagon receptor and decreases the maximal glucagon stimulation observed, with a Kb (concentration of antagonist that shifts the agonist dose-response 2-fold) of 25 nM. These data suggest that L-168,049 is a noncompetitive antagonist of glucagon action. Inclusion of L-168, 049 increases the rate of dissociation of labeled glucagon from the receptor 4-fold, confirming that the compound is a noncompetitive glucagon antagonist. In addition, we have identified two putative transmembrane domain residues, phenylalanine 184 in transmembrane domain 2 and tyrosine 239 in transmembrane domain 3, for which substitution by alanine reduces the affinity of L-168,049 46- and 4. 5-fold, respectively. These mutations do not alter the binding of labeled glucagon, suggesting that the binding sites for glucagon and L-168,049 are distinct.     

Kinetic analysis of the interleukin-13 receptor complex

Interleukin (IL)-13 is a key cytokine associated with the asthmatic phenotype. It signals via its cognate receptor, a complex of IL-13 receptor alpha1 chain (IL-13Ralpha1) with IL-4Ralpha; however, a second protein, IL-13Ralpha2, also binds IL-13. To determine the binding contributions of the individual components of the IL-13 receptor to IL-13, we have employed surface plasmon resonance and equilibrium binding assays to investigate the ligand binding characteristics of shIL-13Ralpha1, shIL-13Ralpha2, and IL-4Ralpha. shIL-13Ralpha1 bound IL-13 with moderate affinity (K(D) = 37.8 +/- 1.8 nm, n = 10), whereas no binding was observed for hIL-4Ralpha. In contrast, shIL-13Ralpha2 produced a high affinity interaction with IL-13 (K(D) = 2.49 +/- 0.94 nm n = 10). IL-13Ralpha2 exhibited the binding characteristics of a negative regulator with a fast association rate and an exceptional slow dissociation rate. Although IL-13 interacted weakly with IL-4Ralpha on its own (K(D) > 50 microm), the presence of hIL-4Ralpha significantly increased the affinity of shIL-13Ralpha1 for IL-13 but had no effect on the binding affinity of IL-13Ralpha2. Detailed kinetic analyses of the binding properties of the heteromeric complexes suggested a sequential mechanism for the binding of IL-13 to its signaling receptor, in which IL-13 first binds to IL-13Ralpha1 and this then recruits IL-4Ralpha to stabilize a high affinity interaction.     

New high affinity H3 receptor agonists without a basic side chain

In this study, we replaced the basic amine function of the known histamine H(3) receptor agonists imbutamine or immepip with non-basic alcohol or hydrocarbon moieties. All compounds in this study show a moderate to high affinity for the cloned human H(3) receptor and, unexpectedly, almost all of them act as potent agonists. Moreover, in the alcohol series, we consistently observed an increased selectivity for the human H(3) receptor over the human H(4) receptor, but none of the compounds in this series possess increased affinity and functional activity compared to their alkylamine congeners. In this new series of compounds VUF5657, 5-(1H-imidazol-4-yl)-pentan-1-ol, is the most potent histamine H(3) receptor agonist (pK(i) = 8.0 and pEC(50) = 8.1) with a 320-fold selectivity at the human H(3) receptor over the human H(4) receptor.     

Design of subtype selective melatonin receptor agonists and antagonists.

Studies of the physiological actions of melatonin have been hindered by the lack of specific, potent and subtype selective agonists and antagonists. In the present study, we describe the utility of a melanophore cell line from Xenopus laevis for exploring structure-activity relationships among novel melatonin analogues and report a novel MT2-selective agonist (IIK7) and MT2-selective receptor antagonist (K185). IIK7 is a potent melatonin receptor agonist in the melanophore model, and in NIH3T3 cells expressing human mt1 and MT2 receptor subtypes. In radioligand binding experiments IIK7 is 90-fold selective for the MT2 subtype. K185 is devoid of agonist activity, but acts as a competitive melatonin antagonist in melanophores. A low concentration (10(-9) M) antagonizes melatonin inhibition of forskolin stimulation of cyclic AMP in NIH3T3 cells expressing human MT2 receptors, but has no effect in cells expressing mt1 receptors. In binding assays, K185 is 140-fold selective for the MT2 subtype.     

Functional consequences of agonist-mediated state transitions in the cholinergic receptor. Studies in cultured muscle cells.

Parameters associated with activation and desensitization of the nicotinic receptor in the BC3H-1 muscle cell line have been compared with the state transitions that result upon combination with agonist. 125I-labeled cobra alpha-toxin is found to bind to an apparent single class of surface nicotinic receptors on the cells in situ with a rate constant of 1.15 x 10(5) M-1 s-1. The competition between cholinergic ligands and alpha-toxin reveals that agonists, but not classical antagonists, will promote a slow conversion to a receptor state where the affinity for agonists is enhanced. Moreover, agonists such as carbamylcholine elicit a permeability increase to 22Na+ ions that slowly decrements at a rate and to an extent closely paralleled by the conversion of the receptor to the high affinity state. Upon removal of the agonist, both the affinity increase and the diminished permeability change are completely reversible and again exhibit similar kinetics for their return to the original state. A comparison of the capacity of full agonists to compete with alpha-toxin binding and elicit a permeability change suggests that in the absence of agonist, receptor predominates in a low affinity activatable state. Binding of agonists to the low affinity state exhibits little if any cooperativity (n = 0.97 to 1.31), while the corresponding permeability change appears more cooperative (n = 1.31 to 1.52). By contrast, when receptors have been previously equilibrated with agonists, occupation of the receptor occurs over a 3- to 5-fold lower concentration range. Binding following equilibration closely correlates with a concomitant decrease in activatable receptor resulting from equivalent exposure to agonist. Furthermore, under equilibrium conditions, the binding of full agonists is typified by a moderate degree of homotropic cooperativity (1.25 to 1.44), enabling the receptor to desensitize over a narrow range of agonist concentration. Simultaneous measurement of occupation and activation parameters has enabled us to compare a state function for desensitization which is generated from binding parameters with the reduction in permeability seen in the desensitization process. A scheme describing the association of agonist with two functionally distinct receptor states is developed to account for the cooperative relationship between agonist binding and desensitization of the receptor.     

Interleukin-1 receptor antagonist binds to the type II interleukin-1 receptor on B cells and neutrophils.

We have examined the binding of human and rodent interleukin-1 receptor antagonist (IL-1ra) to the type II IL-1 receptor on the human B cell line, Raji, on the mouse pre-B cell line, 70Z/3, and on human polymorphonuclear leukocytes (PMNs). Human IL-1ra binds to the receptors on the human B cells with an affinity (KD = 15 +/- 3 nM) equal to that of IL-1 alpha and only 15-fold lower than that of IL-1 beta and, likewise, binds to human PMNs with an affinity (KD = 8 +/- 4 nM) 15-fold lower than that of IL-1 beta. Mouse and rat IL-1ra bind to these two human cell types with an affinity similar to that of the human protein. Human IL-1ra binds very weakly to the type II receptor on the mouse pre-B cells with an affinity (KD = 1.4 +/- 0.2 microM) about 1500-fold lower than human IL-1 beta. Mouse and rat IL-1ra also bind to the mouse pre-B cells with low affinity. The weak binding of the three IL-1ra proteins to these mouse cells appears to be more a consequence of the cell type rather than species specificity. There may be a population of cells for which the actions of IL-1 cannot be effectively opposed by IL-1ra, although this group does not include mature B cells and PMNs.     

Mutations of aspartate 103 in the Hm2 receptor and alterations in receptor binding properties of muscarinic agonists

Aspartate 103 (D103) in the third transmembrane domain of the Hm2 receptor was mutated to glutamate (D103E), asparagine (D103N), or alanine (D103A). As measured by [3H]-NMS, no significant binding was observed in D103A, while a 2-fold decrease in ligand affinity was seen in D103E and a 32-fold decrease in affinity was found in the D103N mutant. Examination of reference agonists showed greater loss of affinity in D103N than in D103E with the rank order of change being: L-607,207>carbachol>arecoline>pilocarpine>oxotremorine>McN-A-343. Of the novel 1-azabicyclo[2.2.1]-heptan-3-one oxime agonists examined, arylacetylene oximes showed little alteration in binding in either the D103E or D103N mutants, while the geometric isomers of several bicyclic aryl-ene-yne oximes showed significant changes in affinity, especially in the D103N mutant. Thus, overall size of the agonist and/or spatial orientation of the molecule within the binding pocket contribute to changes measured in binding.     

Tyrosine 62 of the gamma-aminobutyric acid type A receptor beta 2 subunit is an important determinant of high affinity agonist binding

The gamma-aminobutyric acid type A receptor (GABA(A)R) carries both high (K(D) = 10-30 nm) and low (K(D) = 0.1-1.0 microm) affinity binding sites for agonists. We have used site-directed mutagenesis to identify a specific residue in the rat beta2 subunit that is involved in high affinity agonist binding. Tyrosine residues at positions 62 and 74 were mutated to either phenylalanine or serine and the effects on ligand binding and ion channel activation were investigated after the expression of mutant subunits with wild-type alpha1 and gamma2 subunits in tsA201 cells or in Xenopus oocytes. None of the mutations affected [(3)H]Ro15-4513 binding or impaired allosteric interactions between the low affinity GABA and benzodiazepine sites. Although mutations at position 74 had little effect on [(3)H]muscimol binding, the Y62F mutation decreased the affinity of the high affinity [(3)H]muscimol binding sites by approximately 6-fold, and the Y62S mutation led to a loss of detectable high affinity binding sites. After expression in oocytes, the EC(50) values for both muscimol and GABA-induced activation of Y62F and Y62S receptors were increased by 2- and 6-fold compared with the wild-type. We conclude that Tyr-62 of the beta subunit is an important determinant for high affinity agonist binding to the GABA(A) receptor.     

The loss of an electrostatic contact unique to AMPA receptor ligand binding domain 2 slows channel activation

Ligand-gated ion channels undergo conformational changes that transfer the energy of agonist binding to channel opening. Within ionotropic glutamate receptor (iGluR) subunits, this process is initiated in their bilobate ligand binding domain (LBD) where agonist binding to lobe 1 favors closure of lobe 2 around the agonist and allows formation of interlobe hydrogen bonds. AMPA receptors (GluAs) differ from other iGluRs because glutamate binding causes an aspartate-serine peptide bond in a flexible part of lobe 2 to rotate 180° (flipped conformation), allowing these residues to form cross-cleft H-bonds with tyrosine and glycine in lobe 1. This aspartate also contacts the side chain of a lysine residue in the hydrophobic core of lobe 2 by a salt bridge. We investigated how the peptide flip and electrostatic contact (D655-K660) in GluA3 contribute to receptor function by examining pharmacological and structural properties with an antagonist (CNQX), a partial agonist (kainate), and two full agonists (glutamate and quisqualate) in the wildtype and two mutant receptors. Alanine substitution decreased the agonist potency of GluA3(i)-D655A and GluA3(i)-K660A receptor channels expressed in HEK293 cells and differentially affected agonist binding affinity for isolated LBDs without changing CNQX affinity. Correlations observed in the crystal structures of the mutant LBDs included the loss of the D655-K660 electrostatic contact, agonist-dependent differences in lobe 1 and lobe 2 closure, and unflipped D(A)655-S656 bonds. Glutamate-stimulated activation was slower for both mutants, suggesting that efficient energy transfer of agonist binding within the LBD of AMPA receptors requires an intact tether between the flexible peptide flip domain and the rigid hydrophobic core of lobe 2.     

Temperature-dependent binding of hydrophilic beta-adrenergic receptor ligands to intact human lymphocytes

Important differences in binding characteristics between agonists and antagonists of the beta-adrenergic receptor have been described. However, these observations have been complicated since most available antagonists are much more lipophilic than agonists. In order to separate out those binding characteristics of agonist vs. antagonist from those characteristics of lipophilic vs. hydrophilic ligands, we have studied competition of the hydrophilic ligands isoproterenol (agonist) and CGP-12177 (antagonist) with [125I]iodopindolol binding in intact human lymphocytes. Analyzing competition curves from assays performed at 13 degrees C, 25 degrees C and 37 degrees C we demonstrated that at lower temperatures there was a decrease in IC50 for isoproterenol but not for CGP-12177. Using cells preincubated with isoproterenol then extensively washed, competition curves with both isoproterenol and CGP-12177 were biphasic, and characterized by the appearance of a population of receptors with a low affinity for both hydrophilic ligands. Furthermore, at lower temperatures the biphasic nature of these curves was accentuated and was characterized by a 6-fold and 40-fold increase in the apparent KD of a population of low affinity sites for isoproterenol and CGP-12177, respectively.     

Granulocyte colony-stimulating factor modulation of cytokine receptors on murine bone marrow cells. In vivo and in vitro studies.

Human recombinant granulocyte CSF (G-CSF) modulation of cytokine receptors on murine bone marrow cells (BMC) in vivo and in vitro was investigated. In vivo, G-CSF reduced 125I-G-CSF binding to BMC by greater than 95% within 30 min, with return to base line after 48 h. Human rCSF-1 binding was reduced greater than 85% after 30 min and failed to recover even after 48 h. Murine rTNF-alpha or recombinant granulocyte/macrophage CSF binding was not significantly altered. However, human rIL-1 alpha binding increased greater than 1.5-fold after 3 h, was elevated greater than 5-fold between 6 and 12 h, and declined to base line after 48 h. In vitro, G-CSF induced a greater than 1.5-fold increase in IL-1 binding to BMC after 8 h, suggesting that up-modulation of IL-1 binding in vivo required G-CSF and other influences. Further studies indicated that BMC responded to glucocorticoids and G-CSF with a synergistic increase of IL-1 binding. This synergistic IL-1R modulation was a time- and dose-dependent process and was inhibited by cycloheximide or actinomycin D in a dose-dependent manner. Binding studies further revealed that the synergistic stimulation of IL-1R expression on BMC was probably due to increased receptor number, rather than increased receptor affinity. In addition, this phenomenon was also observed in other hematopoietic cells. Our results demonstrated that G-CSF was capable of stimulating IL-1R expression on BMC both in vivo and in vitro and G-CSF in combination with glucocorticoids synergistically up-modulated IL-1 binding to BMC in vitro. Inasmuch as IL-1 induces the secretion of G-CSF and glucocorticoids in vivo, this synergistic induction may play an important, as yet unknown, role in the inflammatory cascade.