Affinity labeling of cysteine-mutants evidences contact residues in modeled receptor binding sites.

To investigate the topology of binding sites in two ionotropic receptors, we have initiated a strategy combining affinity labeling with cysteine-scanning mutagenesis. For the GABAA receptor we have used reactive derivatives of non-competitive blockers (NCBs) to explore interacting positions in its channel. The polypeptide positions of the M2 segment of the alpha1 subunit which we mutated into cysteine were selected for their established accessibility, as determined by the substituted-cysteine accessibility method (SCAM). Using the Xenopus oocyte expression system, we show that receptors containing mutations V257C and S272C are inactivated by several reactive NCBs. These position-selective inactivations lead to an analysis of NCB binding in the channel. For the NMDA receptor glycine-binding site, the prototype antagonist L-701,324 was derivatized at different positions with different reactive groups. The receptor positions to mutate into cysteine were selected after a 3-D homology model. The observed receptor inactivations are mutant- and probe-selective, leading to an unambiguous chemical docking of the antagonist pharmacophore and supporting the model. The site-specificity of the inactivating reactions is assessed by protection experiments and by mutant to wild-type (WT) comparisons. The scope and limitations of the method are briefly discussed.

Cysteine mutants as chemical sensors for ligand-receptor interactions.

The incorporation of cysteine residues into membrane receptors by mutagenesis has enabled the development of engineered proteins. Chemical modification of the mutant receptor using a wide range of biochemical and biophysical probes has facilitated functional studies of ligand-receptor interactions. In particular, the substituted-cysteine accessibility method (SCAM) represents a successful example of how to probe transmembrane receptor domains after chemical modification of the mutants with sulfydryl-reacting molecules. We propose an extension of this methodology using site-specific affinity probes that react with cysteine mutants to gain reliable structural information on the binding of a ligand in its receptor site.

Evaluation and biological properties of reactive ligands for the mapping of the glycine site on the N-methyl-D-aspartate (NMDA) receptor.

The glycine-binding site of the N-methyl-D-aspartate (NMDA) receptor, given its potential as pharmacological target, has been thoroughly studied by structure-activity relationships, which has made possible its description in terms of spatial limits and interactions of various types. A structural model, based on mutational analysis and sequence alignements, has been proposed. Yet, the amino acid residues responsible for the interactions with the ligand have not been unambiguously characterized. To evidence nucleophilic pocket-lining residues, we have designed and synthesized reactive glycine-site ligands derived from 3-substituted 4-hydroxy-quinolin-2(1H)-ones by introducing various electrophilic groups at different positions of the molecule. These ligands were found to have high affinity at the glycine site and to be functional antagonists by inhibiting glycine/glutamate-induced currents in transfected oocytes. The correlation between their potency and their substitution pattern was strictly consistent with previously established structure-activity relationships. Most ligands displayed intrinsic reactivity toward cysteine, but none inactivated wild-type receptors. This is consistent with the model since it indicates the absence of exposed cysteine in the glycine-binding site. A strategy of cysteine incorporation by point mutations at selected polypeptide positions will create unambiguously localized targets for our reactive probes.

Irreversible site-directed labeling of the 4-aminobutyrate binding site by tritiated meta-sulfonate benzene diazonium. Contribution of a nucleophilic amino acid residue of the alpha1 subunit.

Tritiated meta-sulfonate benzene diazonium ([3H]MSBD), a molecule structurally related to 4-aminobutyrate (GABA), which presents a reactivity toward nucleophilic amino acid residues, was synthesized to investigate the GABA binding site on the GABAA receptor. Irreversible labeling reactions using [3H]MSBD were performed on purified GABAA receptors isolated from cow brain membranes and labeled receptors were analyzed by SDS/PAGE. [3H]MSBD was found to be specifically incorporated into proteins in the 45-60 kDa molecular mass range which were identified as alpha1 subunits and beta2/beta3 subunits by immunoprecipitation with subunit-specific antibodies. The specific immunoprecipitation of alpha and beta subunits confirms that binding of [3H]MSBD occurs at the boundary of these subunits. These labeling results confirm the involvement of nucleophilic residues from the beta subunit but reveal also the contribution of yet unidentified nucleophilic residues on the alpha subunit for the GABA binding site.

Reactive affinity probes for the mapping of the glycine-binding site of the NMDA receptor NR1 subunit.

The glycine co-agonist binding site of the NMDA receptor is a target for the prevention and treatment of neurotoxic and neurodegenerative conditions. Until now, the interactions taking place at this site, and its structure, have been investigated by ligand structure-activity relationships and by site-directed mutagenesis. On the basis of a structural model which is currently proposed for this site, we have designed and synthesized six affinity markers by substituting electrophilic reactive groups in the 4, the 7 and the 3' positions of L 701,324, a high-affinity glycine site antagonist. These compounds compete with 3H-DCKA binding to rat brain membranes at equilibrium with nanomolar to low-micromolar affinities, and antagonize glycine-evoked currents in oocytes transfected with wild-type NR1-NR2B. However, they do not induce a time-shift in binding equilibria, and do not inactivate irreversibly the glycine evoked currents. Since they react only with cysteine at physiological pH, we conclude that there is no such residue in the site, in agreement with the model. Our affinity markers therefore represent potential topological probes for NMDA receptors with sequence positions related to the glycine-binding site mutated into cysteine.

A photoaffinity probe for the polyamine site regulating the NMDA receptor.

Polyamines produce on the NMDA-receptor channel activity a regulatory effect subsequent to their binding to specific sites on the receptor-complex. The photoactivatable polyamine derivative L-azidophenylspermine shows properties which suggest its potential as a photoaffinity probe to investigate the nature and topology of these sites. In the dark, its effect on the binding of tritiated N-(1-[thienyl]cyclohexyl)piperidine ([3H]TCP) to synaptosomal plasma membranes is similar to that of diaminodecane. Arcaine antagonizes both the effects of L-azidophenylspermine and diaminodecane on [3H]TCP binding. L-Azidophenylspermine competes in post-synaptic densities with tritiated spermidine for a unique binding site with an EC50 similar to that of spermine. Upon irradiation, L-azidophenyl-spermine incorporates into this material with a high efficiency to a level consistent with both the Bmax for tritiated spermidine and the estimated density of NMDA receptors in this fraction.

Muscimol and flunitrazepam binding sites in a subcellular fraction enriched in rat cerebellar glomeruli.

In the internal granular layer of the cerebellar cortex the polysynaptic complexes called glomeruli consist mainly of homogeneous populations of glutamatergic and GABAergic synapses, both located on granule cell dendrites. A subcellular fraction enriched in glomeruli was prepared from rat cerebellum, and the distribution of GABAA and of benzodiazepine binding sites between membranes derived from this fraction (fraction G) and from a total cerebellar homogenate (fraction T) was studied. The benzodiazepine and GABA binding sites were measured by the binding of agonists [3H]flunitrazepam and [3H]muscimol, respectively. The results indicate that both binding sites are present, but only slightly enriched, in the glomerular synapses. We found a muscimol/flunitrazepam binding site ratio of two, which is consistent with the enrichment of muscimol binding sites in the granular layer shown by both autoradiographic with radioactive glutamatergic ligands and in situ hybridization experiments respectively.

Identification and characterization of a high-affinity glutamate-controlled TCP binding site in rat brain postsynaptic densities.

The kinetic and equilibrium binding parameters of the phencyclidine receptor ligand [3H]N-[1-(2-thienyl)cyclohexyl]piperidine (TCP) to a postsynaptic density (PSD) subcellular fraction from rat brain were investigated. A single site was found, which was identified as the high-affinity TCP binding site by competition with dibenzocycloalkenimine (MK-801). In contrast, [3H]TCP binds to two sites on the plasma membrane fraction used as a precursor for PSD; on both fractions, [3H]TCP binding responds to glutamate by an increase of the association rate, the dissociation constant and the number of sites being unchanged. In the PSD fraction [3H]3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), an antagonist specific for the N-methyl-D-aspartate (NMDA) site, bound to high- and low-affinity sites. These results ascertain the presence and identity of synaptic NMDA-gated ion channels, which are assumed in the current hypothesis about excitotoxicity, long-term potentiation and learning.

Effect of iron and transferrin on pure oligodendrocytes in culture; characterization of a high-affinity transferrin receptor at different ages.

Oligodendrocytes in pure culture can grow on relatively low iron concentrations (0.1-0.3 microM), in the absence of transferrin; with micromolar concentrations of iron, toxic effects can be seen after one week in culture. When transferrin is added, the toxic effect of iron is increased. These properties account for the mode of selection of oligodendrocytes for pure cultures. Each oligodendrocyte presents between 1100 and 3600 receptor molecules, with a dissociation constant of 0.2-0.6 nM corresponding to a high affinity transferrin-binding site; these constants vary little with age in culture. These receptors may function as autoreceptors regulating transferrin synthesis by oligodendrocytes.

Comparative binding properties of linear and cyclic delta-selective enkephalin analogues: [3H]-[D-Thr2, Leu5] enkephalyl-Thr6 and [3H]-[D-Pen2, D-Pen5] enkephalin.

The range of delta-selectivity of linear and cyclic analogues of enkephalin in rat brain was found to be: [D-Pen2, L-Pen5] enkephalin (DPLPE) greater than [D-Pen2, D-Pen5] enkephalin (DPDPE) greater than [D-Thr2, Leu5] enkephalyl-Thr6 (DTLET) greater than [D-Ser2, Leu5] enkephalyl-Thr6 (DSLET). Saturation experiments performed with [3H]DPDPE and [3H]DTLET in NG108-15 cells and rat brain showed similar binding capacities for both the ligands, but the delta-affinity of [3H]DTLET (KD approximately 1.2 nM) was much better than that of [3H]DPDPE (KD approximately 7.2 nM). The rather low delta-affinity of DPDPE induced high experimental errors cancelling the benefit of its better delta-selectivity. Binding experiments in rat or guinea-pig brains showed, in both cases, the better delta-selectivity of [3H]DTLET compared to [3H]DSLET. The former peptide remains at this time the most appropriate radioactive probe for binding studies of delta-receptor.

A developmentally modified neurone-specific marker in rodent cerebellum.

Out of several monoclonal antibodies secreted by hybridomas resulting from the fusion of a mouse myeloma cell line with spleen cells from mice immunized with cerebellar membranes from 12 day old rats, one, called 11.9, produced an unusual immunolabelling pattern when tested on sections of rat cerebellum. The cerebellar distribution of the antigenic sites recognized by this antibody using an immunoperoxidase technique at the optical and ultrastructural levels is described in detail in this report. The immunoreaction product was found in the adult rat to be associated with the microtubules and the zone immediately beneath the plasma membrane of parallel fibres. In young animals the density of immunostaining appears to be higher than in the adult, and the staining is detectable in addition in the perikaryal cytoplasm of granule cells. Biochemical studies using the Western immunoblot technique demonstrate that the antigens consist of two polypeptides of molecular weights 120 and 185 kD. The possible relation of the antigens to cytoskeletal structures is discussed and the labelling pattern is compared with that produced by other known monoclonal antibodies.

Equilibrium-driven mechanism for preferential adhesion between chick embryo cells.

The experiments presented here confirm the hypothesis according to which, in our experimental system of differential cell adhesion (where we studied the kinetics of the earliest period of adhesion of a suspension of chick embryo neuroblasts to layers of astroblasts or fibroblasts), the mechanism of adhesion appears to consist of two steps, the first of which is a short-term reversible phase corresponding to a binding equilibrium. In fact, adhesion of neuroblasts to each of the two cell layers occurs according to kinetic constants and attains levels which are characteristic for each of the two adhesion systems. In both systems, neuroblasts that have not adhered at equilibrium are able to adhere if inoculated over a fresh cell layer of the same type, as they do during the first inoculation; conversely, neuroblasts that have adhered to a cell layer can be made to de-adhere by substituting cell-free fresh medium to the inoculation medium containing non-adhering neuroblasts. This shows that, as predicted for a reversible equilibrium system, removal of adhering neuroblasts from the system at equilibrium provokes adhesion, and removal of non-adhered neuroblasts provokes de-adhesion. Furthermore the level of adhesion at equilibrium is, in all cases, the same. The reversibility of adhesion, which is almost quantitative during the onset of the equilibrium, gradually decreases with time, indicating the presence of a process of irreversible attachment between cells after the first reversible step. The developmental implications of the complete sequential mechanisms are briefly discussed.

Morphological maturation and survival of chicken and rat embryonic neurons on different culture substrata.

Neuronal cells from chicken and rat embryonic cerebral hemispheres were plated at a low cell concentration and cultured either on collagen or on a supporting continuous glial layer for periods of up to 21 days. The glial layer was either homologous or heterologous with regard to the animal species; the survival and maturation of the neuronal cells in these different conditions were investigated by light and electron microscopy. Neuronal cells cultured on collagen formed aggregates similar to those formed by neuronal cells plated at high cell density as described in a previous paper; a few aggregated neurons formed processes after 24 h and, only after 48 h of culture, more fibres had developed; the majority of the cells progressively degenerated between days 7 and 21 of culture. In contrast to this, neuronal cells cultured on a supporting glial layer, whether homologous or heterologous, progressively differentiated: neuronal perikarya remained well separated from each other and many processes were already formed after 24 h; later on, networks of fibres developed. At the electron microscopic level, microtubules and neurofilaments were present at a high density in the cells and fibres; immature synapses could be found, but infrequently. Differentiated cells were represented mostly by neurons; oligodendroglial cells were absent, and myelinated fibres could not be detected. The highest positive effect on the maturation and survival of neuronal cells was observed in the presence of a layer of glial cells from the same species. These results emphasize the essential role of glial cells for the neuronal maturation in the absence of contact between neuroblasts.

Monoclonal antibodies specific for glial and neuronal antigens in the young rat cerebellum.

Monoclonal antibodies from 15 different hybridomas derived from the fusion of mouse spleen cells with a myeloma cell line were selected. Mice were immunized with the particulate fraction from 10-13-day-old rat cerebella. Hybridoma secreting antibodies were screened simultaneously by both immunocytochemistry and binding assay. Each antibody reacts with specific cerebellar neuronal or glial cells and structures.

Properties of horse liver alcohol dehydrogenase modified by the affinity label 3-chloroacetylpyridine-adenine dinucleotide.

The reactive analogue of NAD+, CPAD+, was incorporated in the horse liver alcohol dehydrogenase (EC 1.1.1.1) linearly with its inactivation, to stoichiometry, with no apparent subunit interaction. No hydride transfer could take place in the modified enzyme, nor the interaction of trans-4-N,N-dimethylaminocinnamaldehyde with its reduced form, indicating an impairment of the accessibility to the catalytic zinc atom. The labeling in the enzyme, alkylated by [carbonyl-14C]CPAD+ was not stable, with a half-life of 32 h.

Inactivation of yeast alcohol dehydrogenase by a reactive coenzyme analogue: 3-chloroacetyl pyridine adenine dinucleotide.

Yeast alcohol dehydrogenase is very rapidly and irreversibly inactivated by 3-chloroacetyl pyridine adenine dinucleotide, a reactive NAD+-analogue (Biellmann et al., 1974, FEBS Lett. 40, 29-32). Kinetic investigations with this compound, and structurally related compounds, show that this inactivation, against which NAD+ provides a complete protection, corresponds to an affinity label. The incorporation of the coenzyme analogue correlates linearly with the enzyme inactivation, the total inactivation corresponding to one mole of inactivator per coenzyme binding site. The pH-dependence of the inactivation rates of the enzyme by this coenzyme analogue and by its reduced form reflects exactly the pH variation of their respective dissociation constants. In spite of a good stability of the label in the non denatured inactivated enzyme, no modified amino-acid residue could be identified. Considering the affinity of this analogue for yeast alcohol dehydrogenase and the strict steric requirements of this enzyme towards its ligands, the nature of the inactivation reaction as well as different possibilities of the loss of the label in the inactivated enzyme are discussed.