Functional reconstitution of the angiotensin II type 2 receptor and G(i) activation.
On the basis of the patterns of conserved amino acid sequence, the angiotensin II type 2 (AT(2)) receptor belongs to the family of serpentine receptors, which relay signals from extracellular stimuli to heterotrimeric G proteins. However, the AT(2) receptor signal transduction mechanisms are poorly understood. We have measured AT(2)-triggered activation of purified heterotrimeric proteins in urea-extracted membranes from cultured COS-7 cells expressing the recombinant receptor. This procedure removes contaminating GTP-binding proteins without inactivating the serpentine receptor. Binding studies using [(125)I] angiotensin (Ang) II revealed a single binding site with a K(d)=0.45 and a capacity of 627 fmol/mg protein in the extracted membranes. The AT(2) receptor caused a rapid activation of alpha(i) and alpha(o) but not of alpha(q) and alpha(s), as measured by radioactive guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding. Activation required the presence of activated receptors, betagamma, and alpha subunits. As a first step aimed at developing an in vitro assay to examine AT(2) receptor pharmacology, we tested a battery of Ang II-related ligands for their ability to promote AT(1) or AT(2) receptor-catalyzed G(i) activation. Two proteolytic fragments of Ang II, Ang III and Ang1-7, also promoted activation of alpha(i) through the AT(2) receptor. Furthermore, we found that [Sar(1),Ala(8)]Ang II is an antagonist for both AT(1) and AT(2) receptors and that CPG42112 behaves as a partial agonist for the AT(2) receptor. In combination with previous observations, these results show that the AT(2) receptor is fully capable of activating G(i) and provides a new tool for exploring AT(2) receptor pharmacology and interactions with G-protein trimers.
Hansen
J L
JL
Laboratory for Molecular Cardiology and the Department of Medicine B, University of Copenhagen, Denmark.
Servant
G
G
Baranski
T J
TJ
Fujita
T
T
Iiri
T
T
Sheikh
S P
SP
D016475:3T3 Cells; D000804:Angiotensin II / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000032:analysis / Q000378:metabolism*; D015870:Gene Expression; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D051379:Mice; D010446:Peptide Fragments / Q000378:metabolism; D017354:Point Mutation; D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014162:Transfection; D014508:Urea
D008024:Ligands; D010446:Peptide Fragments; D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin; D000804:Angiotensin II; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D014508:Urea; D019204:GTP-Binding Proteins