A Region of the Third Intracellular Loop of the Short Form of the D2 Dopamine Receptor Dictates Gi Coupling Specificity

Abstract

The D2 dopamine receptor has two isoforms, the short form (D2s receptor) and the long form (D2l receptor), which differ by the presence of a 29-amino acid insert in the third cytoplasmic loop. Both the D2s and D2l receptors have been shown to couple to members of the Gαi family of G proteins, but whether each isoform couples to specific Gαi protein(s) remains controversial. In previous studies using Gα i mutants resistant to modification by pertussis toxin (Gα iPT), we demonstrated that the D2s receptor couples selectively to Gαi2PT and that the D2l receptor couples selectively to Gαi3PT (Senogles, S. E. (1994) J. Biol. Chem. 269, 23120-23127). In this study, two point mutations of the D2s receptor were created by random mutagenesis (R233G and A234T). The two mutant D2s receptors demonstrated pharmacological characteristics comparable with those of the wild-type D2s receptor, with similar agonist and antagonist binding affinities. We used human embryonic kidney 293 cells stably transfected with Gα i1PT, Gαi2PT, or Gαi3PT to measure agonist-mediated inhibition of forskolin-stimulated cAMP accumulation before and after pertussis toxin treatment. The two mutant D2s receptors demonstrated a change in Gi coupling specificity compared with the wild-type D2s receptor. Whereas the wild-type D2s receptor coupled predominantly to Gαi2PT, mutant R233G coupled preferentially to Gα i3PT, and mutant A234T coupled preferentially to Gα i1PT. These results suggest that this region of the third cytoplasmic loop is crucial for determining Gi protein coupling specificity.