Large scale expression and purification of the rat 5-HT2c receptor.

5-HT2c G-protein coupled receptors located in the central nervous system bind the endogenous neurotransmitters serotonin and couple to G protein to mediate excitatory neurotransmission, which inhibits dopamine and norepinephrine release in the brain. Thus, 5-HT2c receptors play important roles in cognitive function and are potent drug targets. Structural information is needed to elucidate the molecular mechanism of ligand-binding and receptor-activation of the 5-HT2c receptor. Lacking of an efficient expression system that produces sufficient amounts of active and homogenous receptors hinders progress in the functional and structural characterization of the 5-HT2c receptor. We present here a protocol which can be used easily to obtain milligram amount of purified rat 5-HT2c receptors. We established this protocol by protein engineering and optimization of expression and purification based on radioligand-binding assay. The purified and well-characterized rat 5-HT2c receptors are active, stable, homogenous, and ready for 2-dimensional and 3-dimensional crystallization experiments.

4-Bromo-2,5-dimethoxyphenethylamine (2C-B) and structurally related phenylethylamines are potent 5-HT2A receptor antagonists in Xenopus laevis oocytes.

1. We recently described that several 2-(2,5-dimethoxy-4-substituted phenyl)ethylamines (PEAs), including 4-I=2C-I, 4-Br=2C-B, and 4-CH(3)=2C-D analogs, are partial agonists at 5-HT(2C) receptors, and show low or even negligible intrinsic efficacy at 5-HT(2A) receptors. These results raised the proposal that these drugs may act as 5-HT(2) antagonists. 2. To test this hypothesis, Xenopus laevis oocytes were microinjected with the rat clones for 5-HT(2A) or 5-HT(2C) receptors. The above-mentioned PEAs and its 4-H analog (2C-H) blocked the 5-HT-induced currents at 5-HT(2A), but not at the 5-HT(2C) receptor, revealing 5-HT(2) receptor subtype selectivity. The 5-HT(2A) receptor antagonism required a 2-min preincubation to attain maximum inhibition. 3. All PEAs tested shifted the 5-HT concentration-response curves to the right and downward. Their potencies varied with the nature of the C(4) substituent; the relative rank order of their 5-HT(2A) receptor antagonist potency was 2C-I>2C-B>2C-D>2C-H. 4. The present results demonstrate that in X. laevis oocytes, a series of 2,5-dimethoxy-4-substituted PEAs blocked the 5-HT(2A) but not the 5-HT(2C) receptor-mediated responses. As an alternative hypothesis, we suggest that the psychostimulant activity of the PEAs may not be exclusively associated with partial or full 5-HT(2A) receptor agonism.