Fluorescence Resonance Energy Transfer Competitive Binding Assay for Secretin Receptor （Class B-GPCR）

Human secretin is responsible for carrying a number of physiological functions including energy and water homeostasis, thus making secretin receptor a promising target for drug development. For GPCRs （G protein-coupled receptors）, radioactive ligands are usually used in conventional binding assays to characterize the binding affinities of the ligands. An alternative non-hazardous fluorescence based binding assay is lucrative over the radio-ligand assays. Here, we have developed a FRET （fluorescence resonance energy transfer） competitive binding assay for human secretin receptor. The receptor gene sequence is cloned in the SNAP （single nucleotide amplified polymorphisms） tag-plasmid and expressed in CHO （chinese hamster ovary）-K1 cells. Its expression and function is confirmed with immunofluorescence localization and receptor activation. The receptor and the ligand are labeled with fluorescent donor （Tb） and acceptor （Alexa488）. FRET signals are produced when the labeled ligand is bound to the receptor and the same drop when it is displaced by the test compounds. The saturation concentration of the receptor labeling is 100 nM, and the ligand Kd value is 500 nM. At these concentrations, the IC50 of unlabeled secretin is 1.63 4- 3.55 nM. Additionally, few class-B ligands are screened and hold good correlation with traditional radio-ligand assay. Henceforth, this FRET binding assay can be efficiently used as a primary screening tool for peptide analogs.

Characterization of subtype selection properties of R-(-)-DM-phencynonate hydrochloride and its racemate on muscarinic receptors

In order to compare the potential selectivity of R-（-）-DM-phencynonate hydrochloride with its racemate （±）-DM- phencynonate hydrochloride on acetylcholine muscarinic receptor subtypes, the five human acetylcholine muscarinic receptor subtypes （M1- M5） （CHO-hml-5R） were cloned and expressed in Chinese hamster ovary （CHO-K1） cell line. The specific mRNAs of the five acetylcholine muscarinic receptor subtypes were detected by the reverse transcription-polymerase chain reaction （RT-PCR） method, demonstrating the definite expression of muscarinic receptor subtype genes （CHO-hml-5R）. The affinity and saturability of different muscarinic receptor subtypes to [^3H] N-methylscopolamine （[^3H]-NMS） were obtained by radioligand binding assay. Equilibrium binding assay revealed that the maximum binding capacity of [^3H]-NMS （Bmax value） to CHO-hml-5R were 40.22±3.23, 24.53±4.11, 29.65±2.65, 25.41±2.46, 32.78±4.81 pmol/mg·protein, respectively. Kd values of [^3H]-NMS to muscarinic receptors M1 to M5 were 0.97±0.22, 1.16±0.14, 0.99±0.06, 0.56±0.08, 1.12±0.06 nM, respectively. R-（-）-DM- phencynonate hydrochloride was found to block the M4 receptor with a much higher potency （pD2 = 7.48） than those displayed on M1 （pD2 = 6.20）, M2 （pD2 = 5.99）, M3 （pD2 = 5.99） and M5 （pD2 = 6.70） subtypes. However, for （±）-DM-phencynonate hydrochloride, no significant subtype receptor selectivity was found. Both （±）-DM- and R-（-）-DM-phencynonate hydrochloride showed allosteric effects on muscarinic receptors, the Hill coefficient （nH） of five receptor subtypes was less than 1, respectively. The results revealed that R-（-）-DM-phencynonate hydrochloride showed selectivity torwards M4 subtype, and there were allosteric effects for both R-（-）-DM-phencynonate hydrochloride and （±）-DM-phencynonate hydrochloride on muscarinic receptors.

Neuropeptide Y receptors in a protozoa Stylonychia mytilus

Radio ligand binding assays(RLBA) were used to study neuropeptide Y (NPY) receptors in a protozoa Stylonychia mytilus. The experimental results showed that 2-3×10+3/mL Stylonychia cells incubated in Pringsheim solution which contained 3H-NPY could specifically bind 3H-NPY and concomitantly present saturable characteristic. This suggested that Stylonychia possessed some specific binding sites for NPY. Scatchard transformations of binding assay for the NPY receptors at 25℃ are compatible with the specific activity of 42.47 fmol/10+3 cells and the binding equilibrium constant of 0.113 nmol/L. The data of 125I-NPY binding assay to the membrane protein extract of Stylonychia indicated that there was a significant difference between the amount of total bound and nonspecific bound of 125I-NPY. This result indicated that NPY receptors were probably localized mainly on the cell membrane.