I. Recombinant receptors |
A. Evidence for their formation, subunit composition, and stoichiometry, using recombinant receptor expression in heterologous cell systems |
1. The subunit polypeptides must be shown to be expressed |
2. The subunit polypeptides must be shown to coassemble |
a. Coimmunoprecipitation |
b. Physical demonstration of subunit interactions by FRET or similar technique |
c. Formation of pentamers |
d. Unique subunit arrangement, e.g., using concatemers |
3. The corresponding recombinant receptor subtype must be functional |
B. Evidence for unique properties, including pharmacology |
1. Unique biophysical characteristics |
2. Unique pharmacology |
a. Receptor subtype-selective agonists, antagonists, allosteric modulators |
b. Receptor subtype-selective radioligands |
c. Potency and efficacy for a series of ligands |
d. Macrokinetic measures (e.g., apparent EC50 values and binding constants for a series of ligands) |
II. Native receptors |
A. Colocalization of subunits |
1. Tissue colocalization |
2. Cell colocalization (in situ, single-cell RT-PCR) |
3. Subcellular colocalization (light and electron microscopy) |
B. Physical demonstration of subunit interactions (e.g., by coimmunoprecipitation) |
C. Functional demonstration: |
1. Evidence that a given receptor is expressed in real neurons by showing properties (assessed with electrophysiology) corresponding with a recombinant receptor candidate; microscopy may complement |
2. Evidence that a given subunit or subunit combination participates in a specific function in vitro or in vivo using genetically modified mice |