title: Inhibitory GTP-binding regulatory protein Gi3 can couple angiotensin II receptors to inhibition of adenylyl cyclase in hepatocytes.
abstract: Angiotensin II can inhibit hormone-stimulated adenylyl cyclase in intact hepatocytes or in hepatic membrane preparations. Because the response can be blocked by pertussis toxin, the object of the present study was to determine which of the known variants of Gi can couple angiotensin II receptors to inhibition of adenylyl cyclase. The potential candidates were identified by probing RNA isolated from rat hepatocytes with cDNAs specific for the alpha subunits of known toxin-sensitive guanine nucleotide-binding regulatory proteins (G proteins). Hepatocytes contained no detectable RNA for the Go or Gi1 alpha subunits and similar levels of RNA coding for the Gi2 and Gi3 alpha subunits. To determine whether Gi3 could couple angiotensin receptors to inhibition of cyclase, membranes were prepared from hepatocytes whose G proteins were fully ADP-ribosylated with pertussis toxin, and the Gi3 holoprotein purified from rabbit liver was reconstituted into the membranes. The nature of the Gi3 reconstituted into the membrane was assessed by immunoblotting with antibodies specific for the Gi alpha subunits. Reconstitution of 6-10 pmol of Gi3/mg of membrane protein into the toxin-treated membranes restored the ability of 10 nM angiotensin II to inhibit adenylyl cyclase. Because pertussis toxin has nonspecific effects, an assay was developed to measure the interaction of the angiotensin receptor with reconstituted G proteins in normal membranes. In the presence of Mg2+, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) caused a reduction of the affinity of the angiotensin II receptor for 125I-angiotensin II that was stable to washing and the detergents used to reconstitute G proteins into the membranes. Using this protocol to activate G proteins and "uncouple" receptors, the ability of the GDP-liganded form of Gi to restore high affinity binding was examined. Reconstitution of about 10-15 pmol of oligomeric Gi3/mg of membrane protein restored both the high affinity state of the angiotensin II receptor and the ability of GTP gamma S to shift the affinity to a lower state. The same shift in receptor affinity could be accomplished by reconstituting the Gi3 alpha subunit, resolved free of beta gamma subunits, into the membranes. Reconstitution of up to 50 pmol of Gs/mg of membrane protein had no effect on angiotensin II receptor affinity. The results suggest that a major form of Gi in hepatocytes is Gi3 and that it can couple angiotensin receptors to inhibition of adenylyl cyclase.
authors.affiliation: Department of Pharmacology, University of Virginia, Charlottesville 22908.
authors.forename: B F
authors.initials: BF
authors.lastname: Pobiner
authors.forename: J K
authors.initials: JK
authors.lastname: Northup
authors.forename: P H
authors.initials: PH
authors.lastname: Bauer
authors.forename: E D
authors.initials: ED
authors.lastname: Fraser
authors.forename: J C
authors.initials: JC
authors.lastname: Garrison
mesh_terms: D000067956:Adenylyl Cyclase Inhibitors; D000804:Angiotensin II / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D019204:GTP-Binding Proteins / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D008099:Liver / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains; D011945:Receptors, Angiotensin / Q000502:physiology*
chemical_list: D000067956:Adenylyl Cyclase Inhibitors; D012333:RNA, Messenger; D011945:Receptors, Angiotensin; D000804:Angiotensin II; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins; D008274:Magnesium