Reconstitution of functional dopamine D 2s receptor by co-expression of amino- and carboxyl-terminal receptor fragments

Abstract

An N-terminal dopamine D 2s 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 D 2s receptor. The truncated receptor (referred to as D 2trunc) contained transmembrane domains I–V and the N-terminal portion of the third cytoplasmic loop, whereas the C-terminal receptor fragment (referred to as D 2tail) contained transmembrane domains VI and VII and the adjacent intra- and extracellular sequences of the dopamine D 2s receptor. Expression in COS-7 cells of either of these two polypeptides alone did not result in any detectable [ 3H]methylspiperone binding activity. However, specific [ 3H]methylspiperone binding could be observed after coexpression of the D 2trunc and D 2tail 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 D 2s receptor for agonists and antagonists. Functional stimulation of the cotransfected D 2trunc and D 2tail 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 IC 50 of quinpirole was 5.1±0.3 μM. 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.