Prolonged treatment of native mutant (T343K)D 2 SHORT dopamine receptors with the inverse antagonist haloperidol results in an increase in receptor intensity D.J. Franks, B. Gardner1 & P.G. Strange, School of Animal & Microbial Sciences, University of Reading, Reading, RG6 6AJ and 1Pfizer Global R&D, Sandwich, Kent, CT13 9NJ.

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Mutating threonine343 to lysine (T343K) in the C-terminal region of the third intracellular loop of the D_2S dopamine receptor results in a receptor with very low expression. This region is known to be involved in restraining the receptor in its inactive conformation (Kjelsberg et al., 1992), therefore the low expression may be due to the receptor being a constitutively active mutant (CAM). It has been suggested by Smit et al., (1996) that prolonged exposure with inverse agonists can significantly increase the expression of CAMs, therefore this theory was investigated here.

CHO cells stably expressing human D_2S or D_2ST343K dopamine receptors were treated with vehicle or the D₂ inverse agonist haloperidol (Wilson et al., 2001) for 24hours, and then extensively washed to remove any bound ligands before membrane preparation. Saturation binding experiments were performed on membranes (10µg) with [³H]spiperone (5pM-2nM) to determine the number of receptors (B_max) as in Gardner et al., (1996). Statistical significance was determined using an unpaired Student's t test.

Table 1. Saturation analysis of native and mutant D_2S receptors, and the effects of 24hr treatment with an inverse agonist. Data represent mean ± s.e.mean *p<0.002, **p<0.001 (vehicle vs. treatment).

The basal expression levels for D_2S and D_2ST343K receptors were significantly different (Table 1), which indicated some kind of instability of the mutant receptor, either during synthesis or once at the cell surface. Treatment with haloperidol (100nM) for 24hours resulted in a significant increase in the expression of both D_2S (134%) and D_2ST343K (637%) (Table 1). Haloperidol was also shown to have a dose-dependent effect on the expression of D_2ST343K (Fig. 1).

Fig. 1. Effect of increasing haloperidol concentrations on D_2ST343K receptor expression. Data represent mean ± s.e.mean (n3).These data show that haloperidol significantly increases the expression of both D_2S and D_2ST343K dopamine receptors. It is possible that there are two mechanisms of action involved: 1) stabilisation of receptors at the cell surface by promoting their inactive states, 2) assisting with protein folding during synthesis, thereby 'chaperoning' receptors to the cell surface that would otherwise be targeted for degradation.

Kjelsberg M.A. et al., (1992) J. Biol. Chem. 267, 1430-1433.
Smit M.J. et al., (1996) Proc. Natl. Acad. Sci. USA. 93, 6802-6807.
Wilson J. et al., (2001) J. Neurochem. 77, 493-504.
Gardner B. et al., (1996) Br. J. Pharmacol. 118, 1544-1550.

We thank the BBSRC and Pfizer for their financial support.