Reconstitution of functional dopamine D2S receptor by co-expression of amino- and carboxyl- terminal receptor fragments

An N-terminal dopamine D2s receptor clone was constructed and coexpressed in COS-7 cells together with a separate gene fragment coding for the C-terminal sequence of the dopamine D2s receptor. The truncated receptor referred to as D2trunc. contained transmembrane domains I–V and the N-terminal portion of the third cytoplasmic loop, whereas the C-terminal receptor fragment referred to as D2tail. contained transmembrane domains VI and VII and the adjacent intra- and extracellular sequences of the dopamine D2s receptor. w3 Expression in COS-7 cells of either of these two polypeptides alone did not result in any detectable Hxmethylspiperone binding activity. w3 However, specific Hxmethylspiperone binding could be observed after coexpression of the D2trunc and D2tail gene constructs; the number of receptors present on the plasma membrane was about 10% with respect to that of the wild type. The binding properties of the coexpressed fragments were similar to those of the wild-type dopamine D2s receptor for agonists and antagonists. Functional stimulation of the cotransfected D2trunc and D2tail fragments with quinpirole resulted in the inhibition of adenylate cyclase activity. Maximal inhibition corresponds to a 28% decrease in forskolin-stimulated adenylate cyclase. The apparent IC50 of quinpirole was 5.1"0.3 mM. These findings confirm and extend analogous data for other G protein-coupled receptors and indicate that this phenomenon is of general importance for the entire family of these proteins.