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

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-term inal sequence of the dopamine D-2s receptor. The truncated receptor (referr ed to as D-2trunc) contained transmembrane domains I-V and the N-terminal p ortion of the third cytoplasmic loop, whereas the C-terminal receptor fragm ent (referred to as D-2tail) contained transmembrane domains VI and VII and the adjacent intra- and extracellular sequences of the dopamine D-2s recep tor. Expression in COS-7 cells of either of these two polypeptides alone di d not result in any detectable [H-3]methylspiperone binding activity. Howev er, specific [H-3]methylspiperone binding could be observed after coexpress ion of the D-2trunc and D-2tail gene constructs; the number of receptors pr esent 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 ad enylate cyclase. The apparent IC50 of quinpirole was 5.1 +/- 0.3 mu M These findings confirm and extend analogous data for other G protein-coupled rec eptors and indicate that this phenomenon is of general importance for the e ntire family of these proteins. (C) 2000 Elsevier Science B.V. All rights r eserved.