6 research outputs found
Preferential Gs protein coupling of the galanin Gal1 receptor in the μ-opioid-Gal1 receptor heterotetramer
Recent studies have proposed that heteromers of μ-opioid receptors (MORs) and galanin Gal1 receptors (Gal1Rs) localized in the mesencephalon mediate the dopaminergic effects of opioids. The present study reports converging evidence, using a peptide-interfering approach combined with biophysical and biochemical techniques, including total internal reflection fluorescence microscopy, for a predominant homodimeric structure of MOR and Gal1R when expressed individually, and for their preference to form functional heterotetramers when co-expressed. Results show that a heteromerization-dependent change in the Gal1R homodimeric interface leads to a switch in G-protein coupling from inhibitory Gi to stimulatory Gs proteins. The MOR-Gal1R heterotetramer, which is thus bound to Gs via the Gal1R homodimer and Gi via the MOR homodimer, provides the framework for a canonical Gs-Gi antagonist interaction at the adenylyl cyclase level. These novel results shed light on the intense debate about the oligomeric quaternary structure of G protein-coupled receptors, their predilection for heteromer formation, and the resulting functional significance
Development of a Fluorescently Labeled Ligand for Rapid Detection of DAT in Human and Mouse Peripheral Blood Monocytes
Development of a Fluorescently Labeled Ligand for Rapid Detection of DAT in Human and Mouse Peripheral Blood Monocytes
The dopamine transporter (DAT) is one of the key regulators
of
dopamine (DA) signaling in the central nervous system (CNS) and in
the periphery. Recent reports in a model of Parkinson’s disease
(PD) have shown that dopamine neuronal loss in the CNS impacts the
expression of DAT in peripheral immune cells. The mechanism underlying
this connection is still unclear but could be illuminated with sensitive
and high-throughput detection of DAT-expressing immune cells in the
circulation. Herein, we have developed fluorescently labeled ligands
(FLL) that bind to surface-expressing DAT with high affinity and selectivity.
The diSulfoCy5-FLL (GC04-38) was utilized to label DAT
in human and mouse peripheral blood mononuclear cells (PBMCs) that
were analyzed via flow cytometry. Selective labeling was validated
using DAT KO mouse PBMCs. Our studies provide an efficient and highly
sensitive method using this novel DAT-selective FLL to advance our
fundamental understanding of DAT expression and activity in PBMCs
in health and disease and as a potential peripheral biomarker
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Synthesis, Pharmacological Characterization, and Structure–Activity Relationships of Noncanonical Selective Agonists for α7 nAChRs
A lack of selectivity of classical agonists for the nicotinic acetylcholine receptors (nAChR) has prompted us to identify and develop a distinct scaffold of α7 nAChR-selective ligands. Noncanonical 2,4,6-substituted pyrimidine analogues were framed around compound 40 for a structure-activity relationship study. The new lead compounds activate selectively the α7 nAChRs with EC50's between 30 and 140 nM in a PNU-120596-dependent, cell-based calcium influx assay. After characterizing the expanded lead landscape, we ranked the compounds for rapid activation using Xenopus oocytes expressing human α7 nAChR with a two-electrode voltage clamp. This approach enabled us to define the molecular determinants governing rapid activation, agonist potency, and desensitization of α7 nAChRs after exposure to pyrimidine analogues, thereby distinguishing this subclass of noncanonical agonists from previously defined types of agonists (agonists, partial agonists, silent agonists, and ago-PAMs). By NMR, we analyzed pKa values for ionization of lead candidates, demonstrating distinctive modes of interaction for this landscape of ligands
NOVEL COMPOUNDS TARGETING SYNAPSIN III RESCUE ALPHA-SYNUCLEIN AGGREGATION AND NIGROSTRIATAL DYSFUNCTION AND DEGENERATION IN EXPERIMENTAL MODELS OF PARKINSON’S DISEASE
Substituted 2‑Aminopyrimidines Selective for α7-Nicotinic Acetylcholine Receptor Activation and Association with Acetylcholine Binding Proteins
Through studies with
ligand binding to the acetylcholine binding
protein (AChBP), we previously identified a series of 4,6-substituted
2-aminopyrimidines that associate with this soluble surrogate of the
nicotinic acetylcholine receptor (nAChR) in a cooperative fashion,
not seen for classical nicotinic agonists and antagonists. To examine
receptor interactions of this structural family on ligand-gated ion
channels, we employed HEK cells transfected with cDNAs encoding three
requisite receptor subtypes: α7-nAChR, α4β2-nAChR,
and a serotonin receptor (5-HT<sub>3A</sub>R), along with a fluorescent
reporter. Initial screening of a series of over 50 newly characterized
2-aminopyrimidines with affinity for AChBP showed only two to be agonists
on the α7-nAChR below 10 μM concentration. Their unique
structural features were incorporated into design of a second subset
of 2-aminopyrimidines yielding several congeners that elicited α7
activation with EC<sub>50</sub> values of 70 nM and <i>K</i><sub>d</sub> values for AChBP in a similar range. Several compounds
within this series exhibit specificity for the α7-nAChR, showing
no activation or antagonism of α4β2-nAChR or 5-HT3AR at
concentrations up to 10 μM, while others were weaker antagonists
(or partial agonists) on these receptors. Analysis following cocrystallization
of four ligand complexes with AChBP show binding at the subunit interface,
but with an orientation or binding pose that differs from classical
nicotinic agonists and antagonists and from the previously analyzed
set of 2-aminopyrimidines that displayed distinct cooperative interactions
with AChBP. Orientations of aromatic side chains of these complexes
are distinctive, suggesting new modes of binding at the agonist-antagonist
site and perhaps an allosteric action for heteromeric nAChRs