A region of the third intracellular loop of the short form of the D2 dopamine receptor dictates Gi coupling specificity.
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 G alpha(i) family of G proteins, but whether each isoform couples to specific G alpha(i) protein(s) remains controversial. In previous studies using G alpha(i) mutants resistant to modification by pertussis toxin (G alpha(i)PT), we demonstrated that the D2s receptor couples selectively to G alpha(i2)PT and that the D2l receptor couples selectively to G alpha(i3)PT (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 G alpha(i1)PT, G alpha(i2)PT, or G alpha(i3)PT 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 G(i) coupling specificity compared with the wild-type D2s receptor. Whereas the wild-type D2s receptor coupled predominantly to G alpha(i2)PT, mutant R233G coupled preferentially to G alpha(i3)PT, and mutant A234T coupled preferentially to G alpha(i1)PT. These results suggest that this region of the third cytoplasmic loop is crucial for determining G(i) protein coupling specificity.
Department of Molecular Sciences, College of Medicine, University of Tennessee Health Science Center, 858 Madison Avenue, Suite G01, Memphis, TN 38163, USA. ssenogles@utmem.edu
Susan E
SE
Senogles
Tamra L
TL
Heimert
Emilia Riviera
ER
Odife
Michael W
MW
Quasney
D000595:Amino Acid Sequence; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D037342:Pertussis Toxin / Q000494:pharmacology; D017354:Point Mutation; D017448:Receptors, Dopamine D2 / Q000737:chemistry* / Q000502:physiology
D017448:Receptors, Dopamine D2; D000242:Cyclic AMP; D037342:Pertussis Toxin; D019206:GTP-Binding Protein alpha Subunits, Gi-Go