Multiple Gi protein subtypes regulate a single effector mechanism.
alpha 2-Adrenergic receptor (alpha 2-AR) responses are mediated by the pertussis toxin-sensitive guanine nucleotide-binding protein (G protein) Gi. Because all three known Gi subtypes are inactivated by pertussis toxin, it has been difficult to determine which of the subtypes are involved in alpha 2-AR responses. In order to investigate alpha 2-AR/Gi coupling, we performed binding and adenylyl cyclase experiments in membranes from CHO-K1 cells transfected with the human alpha 2A-AR. Antisera directed against the carboxyl-terminal region of the Gi1/Gi2 or the Gi3 proteins were used to determine which subtypes were important for high affinity agonist binding and inhibition of adenylyl cyclase. The CHO-K1 cell membranes exhibited immunoreactivity at an apparent molecular mass of 40-41 kDa for both Gi1/Gi2 and Gi3 antisera. Western blot analysis, using purified bovine brain G proteins for comparison, demonstrated that the transfected CHO-K1 cells possess Gi2 and Gi3. High affinity guanosine 5'-(beta,gamma-imido) triphosphate-sensitive binding of the alpha 2-AR agonists [3H]bromoxidine and p-[125I]iodoclonidine ([125I]PIC) was reduced by 30-50% by either the Gi1/Gi2 or Gi3 antiserum. Bromoxidine (1 microM) and PIC (1 microM) inhibited membrane adenylyl cyclase by 34 and 27%, respectively. Gi3 antiserum reduced the inhibition by 26% and 67% for bromoxidine and PIC, respectively. The Gi1/Gi2 antiserum reduced the inhibition by 56% and 63% for bromoxidine and PIC, respectively. Furthermore, when both antisera were used together, there was a complete reversal of alpha 2-AR-mediated inhibition. These observations provide evidence of alpha 2A-AR coupling to at least two subtypes of Gi proteins and the first evidence of functional involvement of Gi3 in the inhibition of adenylyl cyclase.
Gerhardt
M A
MA
Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109-0626.
Neubig
R R
RR
D000262:Adenylyl Cyclases / Q000032:analysis; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D019204:GTP-Binding Proteins / Q000145:classification / Q000276:immunology / Q000502:physiology*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D007106:Immune Sera / Q000276:immunology; D011942:Receptors, Adrenergic, alpha / Q000502:physiology*
D007106:Immune Sera; D011942:Receptors, Adrenergic, alpha; D006165:Guanylyl Imidodiphosphate; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases