The D2 dopamine receptor isoforms signal through distinct Gi alpha proteins to inhibit adenylyl cyclase. A study with site-directed mutant Gi alpha proteins.
Site-directed mutations of the cDNA for Gi1 alpha, Gi21 alpha, and Gi3 alpha were constructed which changed the cysteine residue at the C terminus to a glycine residue (Gi alpha PT). This mutation of the Gi alpha would not permit the subsequent covalent modification by pertussis toxin, which requires the cysteine moiety. The cDNA for each of the mutant Gi alpha subunits was transfected into GH4C1 cells with either of the alternative splice forms of the D2 dopamine receptor and clonal lines were generated. After treatment with pertussis toxin to remove the contribution from endogenous Gi proteins, the receptor-mediated inhibition of adenylyl cyclase was examined. The D2 dopamine receptor, short form (D2s) signaled through the Gi2 alpha PT mutant in these cells with an affinity for agonist which was comparable to that observed in cells transfected with the cDNA for D2s alone or the signaling observed in the absence of pertussis toxin. The long form of the D2 dopamine receptor (D2l) signaled through the Gi3 alpha PT mutant to inhibit forskolin-stimulated adenylyl cyclase, with an affinity for agonist comparable to that observed in cells transfected with the cDNA for D2l alone. The receptor for somatostatin (somatotropin release inhibiting factor) was used as an endogenous control receptor in these cell lines. The somatotropin release inhibiting factor was able to signal through both Gi1 alpha PT and Gi3 alpha PT to inhibit forskolin-stimulated adenylyl cyclase. These results indicated that receptors use distinct Gi proteins to signal to a common effector.
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Department of Biochemistry, University of Tennessee, Memphis 38163.
D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000187:drug effects; D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism; D018076:DNA, Complementary; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D037342:Pertussis Toxin; D051381:Rats; D017448:Receptors, Dopamine D2 / Q000378:metabolism*; D015398:Signal Transduction; D013004:Somatostatin / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D018076:DNA, Complementary; D017448:Receptors, Dopamine D2; D010566:Virulence Factors, Bordetella; D013004:Somatostatin; D000242:Cyclic AMP; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases