G-protein-coupled receptor oligomers: two or more for what? Lessons from mGlu and GABAB receptors.

G-protein-coupled receptors (GPCRs) are key players in the precise tuning of intercellullar communication. In the brain, both major neurotransmitters, glutamate and GABA, act on specific GPCRs [the metabotropic glutamate (mGlu) and GABA(B) receptors] to modulate synaptic transmission. These receptors are encoded by the largest gene family, and have been found to associate into both homo- and hetero-oligomers, which increases the complexity of this cell communication system. Here we show that dimerization is required for mGlu and GABA(B) receptors to function, since the activation process requires a relative movement between the subunits to occur. We will also show that, in contrast to the mGlu receptors, which form strict dimers, the GABA(B) receptors assemble into larger complexes, both in transfected cells and in the brain, resulting in a decreased G-protein coupling efficacy. We propose that GABA(B) receptor oligomerization offers a way to increase the possibility of modulating receptor signalling and activity, allowing the same receptor protein to have specific properties in neurons at different locations.

Time resolved amplification of cryptate emission: a versatile technology to trace biomolecular interactions.

Fluorescence resonance energy transfer (FRET) in association with a time-resolved fluorescence mode of detection was used to design a new homogeneous technology suitable to monitor biomolecular interactions. A lanthanide cryptate characterised by a long lived fluorescence emission was used as donor and a cross-linked allophycocyanine was used as acceptor. This new donor/acceptor pair displayed an exceptionally large Forster radius of 9 nm. This allowed to build up a set of labelling strategies to probe the interactions between biomolecules with an emphasis on fully indirect cassette formats particularly suitable for high throughput screening applications. Herein we describe the basics of the technology, review the latest applications to the study of molecular interactions involved in cells and new oligonucleotides based assays.

Homogeneous time resolved fluorescence resonance energy transfer using rare earth cryptates as a tool for probing molecular interactions in biology.

A homogeneous assay technology using time resolved fluorescence and fluorescence resonance energy transfer is described. A new class of fluorescent complexes, the cryptates, have been used as fluorescent donor with cross-linked allophycocyanin as acceptor. This new donor/acceptor shows an exceptionally high Förster distance R0 of 9 nm. This allows to build up a set of strategies to probe the interactions of biomolecules in biology, particularly for high throughput screening applications. In this article, we describe the basics of the technology and review applications developed for studying different key molecular interactions involved in cellular processes.

Allophycocyanin 1 as a near-infrared fluorescent tracer: isolation, characterization, chemical modification, and use in a homogeneous fluorescence resonance energy transfer system.

Allophycocyanin 1 (APC1), isolated from Mastigocladus laminosus, retains the same (alpha-beta)(3) trimeric structure as allophycocyanin (APC), but incorporates a peptide linker in its core leading to a 28% increase in its fluorescence quantum yield compared to APC. Moreover, APC1 exhibits an unexpectedly good stability at very low concentrations, at extreme pHs, or diluted in a low ionic strength medium whereas, under the same conditions, APC dissociates into an (alpha-beta) monomer, indicating that the peptide linker acts as a stabilizer of its trimeric structure. APC1 crosslinking experiments performed using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide gave a high reaction yield (95%) and showed a similar crosslinking process as previously described for APC. Fluorescence quantum yields of crosslinked APC1 or APC decrease by 20% after labeling on antibody or streptavidin. However, quantum yields of the crosslinked APC1 conjugates remain 25% higher than those of crosslinked APC conjugates. Associated with a europium trisbipyridine cryptate as donor, crosslinked APC1 was compared with crosslinked APC as acceptor in homogeneous time resolved fluorescence technology based on a fluorescence resonance energy transfer process. Using crosslinked APC1, assay performances were increased by 20%, showing that APC1 could be considered as a very promising near infrared fluorescent probe to replace APC in its biological applications.