Time-resolved FRET between GPCR ligands reveals oligomers in native tissues.

G protein-coupled receptor (GPCR) oligomers have been proposed to play critical roles in cell signaling, but confirmation of their existence in a native context remains elusive, as no direct interactions between receptors have been reported. To demonstrate their presence in native tissues, we developed a time-resolved FRET strategy that is based on receptor labeling with selective fluorescent ligands. Specific FRET signals were observed with four different receptors expressed in cell lines, consistent with their dimeric or oligomeric nature in these transfected cells.

Structure of the third intracellular loop of the vasopressin V2 receptor and conformational changes upon binding to gC1qR.

The V2 vasopressin receptor is a G-protein-coupled receptor that regulates the renal antidiuretic response. Its third intracellular loop is involved in the coupling not only with the GalphaS protein but also with gC1qR, a potential chaperone of G-protein-coupled receptors. In this report, we describe the NMR solution structure of the V2 i3 loop under a cyclized form (i3_cyc) and characterize its interaction with gC1qR.

Toward efficient drug screening by homogeneous assays based on the development of new fluorescent vasopressin and oxytocin receptor ligands.

A series of fluorescent ligands designed for vasopressin and oxytocin G protein-coupled receptors was synthesized and characterized to develop fluorescence polarization or homogeneous time-resolved fluorescence (HTRF) binding assays. These ligands, labeled with europium pyridine-bis-bipyridine cryptate or with Alexa 488,546,647 selectively bound to the vasopressin V1a and oxytocin receptors with high affinities and exhibited antagonistic properties. The affinities of several unlabeled ligands determined by our homogeneous assays on membrane preparations or on intact cells into 96- and 384-well plate formats were similar to those determined by usual radioligand binding methods.

Design of benzophenone-containing photoactivatable linear vasopressin antagonists: pharmacological and photoreactive properties.

We designed and synthesized new photoactivatable linear vasopressin analogues containing benzophenone photophores. All compounds were monitored and purified using RP-HPLC and characterized by mass spectrometry. Affinity and selectivity were determined in CHO cells expressing either human V(1a), V(1b) or V(2) receptor subtypes.