TABLE 2

Criteria for inclusion on a list of native receptor subtypes

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