Mary McVey

Monitoring Receptor Oligomerization Using Time-resolved Fluorescence Resonance Energy Transfer and Bioluminescence Resonance Energy Transfer THE HUMAN δ-OPIOID RECEPTOR DISPLAYS CONSTITUTIVE OLIGOMERIZATION AT THE CELL SURFACE, WHICH IS NOT REGULATED BY RECEPTOR OCCUPANCY

Oligomerization of the human δ-opioid receptor and its regulation by ligand occupancy were explored following expression in HEK293 cells using each of co-immunoprecipitation of differentially epitope-tagged forms of the receptor, bioluminescence resonance energy transfer and time-resolved fluorescence resonance energy transfer. All of the approaches identified constitutively formed receptor oligomers, and the time-resolved fluorescence studies confirmed the presence of such homo-oligomers at the cell surface. Neither the agonist ligand [d-Ala2,d-Leu5]enkephalin nor the inverse agonist ligand ICI174864 were able to modulate the oligomerization status of this receptor. Interactions between co-expressed δ-opioid receptors and β2-adrenoreceptors were observed in co-immunoprecipitation studies. Such hetero-oligomers could also be detected using bioluminescence resonance energy transfer although the signal obtained was substantially smaller than for homo-oligomers of either receptor type. Signal corresponding to the δ-opioid receptor-β2-adrenoreceptor hetero-oligomer was increased in the presence of agonist for either receptor. However, substantial levels of this hetero-oligomer were not detected at the cell surface using time-resolved fluorescence resonance energy transfer. These studies demonstrate that, following transient transfection of HEK293 cells, constitutively formed oligomers of the human δ-opioid receptor can be detected by a variety of approaches. However, these are not regulated by ligand occupancy. They also indicate that time-resolved fluorescence resonance energy transfer represents a means to detect such oligomers at the cell surface in populations of intact cells.

Homo- and hetero-oligomeric interactions between G-protein-coupled receptors in living cells monitored by two variants of bioluminescence resonance energy transfer (BRET): hetero-oligomers between receptor subtypes form more efficiently than between less closely related sequences.

Homo- and hetero-oligomerization of G-protein-coupled receptors (GPCRs) were examined in HEK-293 cells using two variants of bioluminescence resonance energy transfer (BRET). BRET(2) (a variant of BRET) offers greatly improved separation of the emission spectra of the donor and acceptor moieties compared with traditional BRET. Previously recorded homo-oligomerization of the human delta-opioid receptor was confirmed using BRET(2). Homo-oligomerization of the kappa-opioid receptor was observed using both BRET techniques. Both homo- and hetero-oligomers, containing both delta- and kappa-opioid receptors, were unaffected by the presence of receptor ligands. BRET detection of opioid receptor homo- and hetero-oligomers required expression of 50,000-100,000 copies of the receptor energy acceptor construct per cell. The effectiveness of delta-kappa-opioid receptor hetero-oligomer formation was as great as for homomeric interactions. The capacity of the two opioid receptors to form oligomeric complexes with the beta(2)-adrenoceptor was also assessed. Although such interactions were detected, at least 250,000 copies per cell of the energy acceptor were required. Requirement for high levels of receptor expression was equally pronounced in attempts to measure hetero-oligomer formation between the kappa-opioid receptor and the thyrotropin-releasing hormone receptor-1. These studies indicate that constitutively formed homo- and hetero-oligomers of opioid receptor subtypes can be detected in living cells containing less than 100,000 copies of the receptors. However, although hetero-oligomeric interactions between certain less closely related GPCRs can be detected, they appear to be of lower affinity than homo- or hetero-oligomers containing closely related sequences. Interactions recorded between certain GPCR family members in heterologous expression systems are likely to be artefacts of extreme levels of overexpression.

A comparative analysis of the neuroprotective properties of competitive and uncompetitive n-methyl-d-aspartate receptor antagonists in vivo: Implications for the process of excitotoxic degeneration and its therapy

Injection of the N-methyl-D-aspartate receptor agonist, quinolinic acid, into the rat striatum in vivo results in the degeneration of cholinergic and GABAergic neurons, as determined seven days later using the marker enzymes, choline acetyltransferase and glutamate decarboxylase, respectively. Such damage was dose-dependently prevented by CGP 37849 or MK-801 (competitive and uncompetitive N-methyl-D-aspartate receptor antagonists, respectively) administered systemically or intrastriatally at the same time as quinolinic acid. The neuroprotective activity of CGP 37849 was associated with the D-enantiomer, CGP 40116 (ED50 7.5 mg/kg i.p.), which was approximately 1.5-fold and 3.5-fold more potent than the related compounds, D-CPPene and CGS 19755, respectively. CGP 37849 was a weaker neuroprotectant than MK-801 (ED50 0.8 mg/kg i.p) when administered systemically, but was dramatically more potent following coinjection with quinolinic acid (ED50s 0.2 and 117 nmol, respectively). When injected intrastriatally 0.5-2 h post-quinolinic acid, CGP 37849 was protective over the entire period studied, whereas MK-801 was less effective at all post-quinolinic acid injection times. The finding that CGP 37849 is neuroprotective when administered intrastriatally 1-2 h post-quinolinic acid supports the hypothesis that a period exists following excitotoxic insult in which neurons are not committed to die, and can be rescued by blockade of ongoing N-methyl-D-aspartate receptor activation. Competition studies indicated that, when coinjected with 100-400 nmol quinolinic acid into the striatum, CGP 37849 exhibited kinetics predicted of a competitive N-methyl-D-aspartate receptor antagonist (declining neuroprotective potency with increasing doses of agonist), whereas MK-801 displayed a complex picture, with weak protective activity at low doses of quinolinic acid. Following systemic administration, neither antagonist was markedly affected by the dose of excitotoxin. When given i.p. at up to 6 h post-quinolinic acid, CGP 37849 and MK-801 showed essentially identical profiles of post-insult protection; degeneration of cholinergic neurons was reduced significantly throughout the entire post-insult period, whereas GABAergic neurons were protected only when drugs were administered 2 h or earlier post-quinolinic acid. The data indicate that competitive and uncompetitive N-methyl-D-aspartate receptor antagonists are effective neuroprotectants in vivo, and that parameters such as drug lipophilicity or mechanism of action at the receptor do not impinge upon their properties as systemically active cerebroprotectants.

EFFECTS OF SUBSTRATE AND INHIBITOR BINDING ON PROTEOLYSIS OF ISOLEUCYL-TRNA SYNTHETASE FROM STAPHYLOCOCCUS AUREUS

Binding of ligands to isoleucyl-tRNA synthetase (IleRS; E) from Staphylococcus aureus was investigated through effects on proteolytic digestion. Approximately 50-fold higher concentrations of protease (trypsin or chymotrypsin) were required to inactivate IleRS after incubation with substrates and formation of the E·Ile-AMP intermediate compared with free E. Binding of pseudomonic acid A (PS-A) or isoleucynol adenylate (Ile-ol-AMP) also induced resistance to proteolysis and altered the patterns of IleRS cleavage fragments in an inhibitor-class specific manner. The determinants for PS-A binding were investigated via proteolysis of E·[3H]PS-A. Limited proteolysis of E·[3H]PS-A (excising residues 186–407) could be achieved without significant loss of bound inhibitor, eliminating this region as contributing to inhibitor binding. Assays were developed which allowed IleRS proteolysis to be readily followed using fluorescence polarization. Inhibitor-protected IleRS was labeled with fluorescein isothiocyanate with only a small effect upon catalytic activity (Fl-IleRS). The (pseudo) kinetics of proteolytic cleavage of Fl-IleRS could be measured at low nanomolar Fl-IleRS concentrations in 96/384-well microtiter plates, allowing real-time monitoring of dose-dependent protection from proteolysis. Thus, inhibitor (and substrate) binding could be reproducibly assessed in the absence of measurements of catalytic acitvity. This could potentially form the basis of novel screening assays for ligands to other proteins.