Fluorescent ligands for studying neuropeptide receptors by confocal microscopy

Beaudet, Alain; Nouel, Dominique; Stroh, Thomas; Vandenbulcke, Franck; Dal-Farra, C.; Vincent, JP

doi:10.1590/s0100-879x1998001100017

Cited by 35 publications

(12 citation statements)

References 41 publications

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“…[43] K D dependence on the choice of fluorophores has previously been observed for other fluorescent ligand derivatives as well. [44] We also studied binding of a known inverse agonist, SR48692, to NTS1B and obtained a K D of 15±11 nM using label-free MST (Fig. 5D).…”

Section: Resultsmentioning

confidence: 99%

Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions

Seidel

Dijkman²,

Lea³

et al. 2013

Methods

541

478

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Microscale thermophoresis (MST) allows for quantitative analysis of protein interactions in free solution and with low sample consumption. The technique is based on thermophoresis, the directed motion of molecules in temperature gradients. Thermophoresis is highly sensitive to all types of binding-induced changes of molecular properties, be it in size, charge, hydration shell or conformation. In an all-optical approach, an infrared laser is used for local heating, and molecule mobility in the temperature gradient is analyzed via fluorescence. In standard MST one binding partner is fluorescently labeled. However, MST can also be performed label-free by exploiting intrinsic protein UV-fluorescence. Despite the high molecular weight ratio, the interaction of small molecules and peptides with proteins is readily accessible by MST. Furthermore, MST assays are highly adaptable to fit to the diverse requirements of different biomolecules, such as membrane proteins to be stabilized in solution. The type of buffer and additives can be chosen freely. Measuring is even possible in complex bioliquids like cell lysate allowing close to in vivo conditions without sample purification. Binding modes that are quantifiable via MST include dimerization, cooperativity and competition. Thus, its flexibility in assay design qualifies MST for analysis of biomolecular interactions in complex experimental settings, which we herein demonstrate by addressing typically challenging types of binding events from various fields of life science.

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Section: Resultsmentioning

confidence: 99%

Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions

Seidel

Dijkman²,

Lea³

et al. 2013

Methods

541

478

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“…Fluorescent peptide ligands, in conjunction with confocal microscopy, have been used previously to study binding and internalization of G protein-coupled receptors (Beaudet et al, 1998). In the specific case of the opioid receptors the development of receptor antibodies, Flag-tagged antibodies for epitope-tagged receptors and fluorescent peptides have been reported (Kolb et al, 1983;Arden et al, 1995;Keith et al 1996;Sternini et al, 1996;Gaudriault et al, 1997;Whistler et al, 1999).…”

Section: Discussionmentioning

confidence: 99%

Binding and Internalization of Fluorescent Opioid Peptide Conjugates in Living Cells

Arttamangkul

Alvarez-Maubecin

Thomas³

et al. 2000

Mol Pharmacol

108

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The dynamics of agonist-stimulated opioid receptor internalization and trafficking have been difficult to study in living cells in part because the available probes were inadequate. To overcome this obstacle, six new fluorescent opioid peptides were developed. Dermorphin (DERM), deltorphin (DELT), TIPP, and endomorphin were conjugated to BODIPY TR or Alexa Fluor 488, two fluorescent dyes with distinct hydrophobic properties. In membrane binding assays the fluorescent conjugates DERM-A488 or -BTR, DELT-A488 or -BTR, and TIPP-A488 displayed good binding affinity and selectivity for mu- and delta-opioid receptor subtypes. Furthermore, the fluorescent conjugates of dermorphin and deltorphin were biologically active as demonstrated by their ability to hyperpolarize locus coeruleus neurons (DERM-A488 or -BTR) and inhibit calcium currents in NG108-15 (DELT-A488). Both of these responses were antagonized by naloxone. In conjunction with confocal fluorescent microscopy the trafficking of these fluorescent ligands was monitored in real-time. The internalization of these ligands by mu- and delta-opioid receptors was found to be naloxone-sensitive and temperature-dependent. Interestingly, once these ligands were internalized the fluorescent puncta that formed became distributed in one of two patterns. In Chinese hamster ovary cells heterologously expressing either mu- or delta-opioid receptors the intracellular puncta were concentrated in the perinuclear region of the cell, whereas they were distributed throughout the cytoplasm in cells derived from either NG108-15 or SH-SY5Y cells. In summary, we have demonstrated that these novel, fluorescent opioid peptide conjugates permit real-time visual tracking of receptor-ligand complexes, including their internalization and trafficking, in living cells.

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“…In some experiments, acid wash stripping (pH 2.0) of the primary antibodies was performed before proceeding with fixation to visualize specifically endocytosed receptors (Mazella et al ., 1991; Beaudet et al ., 1998; Fourgeaud et al ., 2004).…”

Section: Methodsmentioning

confidence: 99%

Dissociation and trafficking of rat GABAB receptor heterodimer upon chronic capsaicin stimulation

Laffray¹,

Tan²,

Dulluc³

et al. 2007

Eur J of Neuroscience

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Gamma-aminobutyric acid type B receptors (GABAB) are G-protein-coupled receptors that mediate GABAergic inhibition in the brain. Their functional expression is dependent upon the formation of heterodimers between GABAB1 and GABAB2 subunits, a process that occurs within the endoplasmic reticulum. However, the mechanisms that regulate GABAB receptor oligomerization at the plasma membrane remain largely unknown. We first characterized the functional cytoarchitecture of an organotypic co-culture model of rat dorsal root ganglia and spinal cord. Subsequently, we studied the interactions between GABAB subunits after chronic stimulation of sensory fibres with capsaicin. Surface labelling of recombinant proteins showed a decrease in subunit co-localization and GABAB2 labelling, after capsaicin treatment. In these conditions, fluorescence lifetime imaging measurements further demonstrated a loss of interactions between green fluorescent protein-GABAB1b and t-dimer discosoma sp red fluorescent protein-GABAB2 subunits. Finally, we established that the GABAB receptor undergoes clathrin-dependent internalization and rapid recycling to the plasma membrane following activation with baclofen, a GABAB agonist. However, in cultures chronically stimulated with capsaicin, the agonist-induced endocytosis was decreased, reflecting changes in the dimeric state of the receptor. Taken together, our results indicate that the chronic stimulation of sensory fibres can dissociate the GABAB heterodimer and alters its responsiveness to the endogenous ligand. Chronic stimulation thus modulates receptor oligomerization, providing additional levels of control of signalling.

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Fluorescent ligands for studying neuropeptide receptors by confocal microscopy

Cited by 35 publications

References 41 publications

Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions

Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions

Binding and Internalization of Fluorescent Opioid Peptide Conjugates in Living Cells

Dissociation and trafficking of rat GABAB receptor heterodimer upon chronic capsaicin stimulation

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