Constitutive activity of human angiotensin II type-1 receptors by Gq overexpression.
We have developed an inducible HEK293/Tet-On cell line that transiently expresses both FLAG-tagged human angiotensin II type-I receptors (FLAG-hAT(1)R) and G(q)alpha G protein subunits in response to doxycycline. High and tightly regulated levels of FLAG-hAT(1)R (740+/-57 fmol/mg protein) and G(q)alpha (36-fold increase compared with non-induced cells) overexpression were consistently achieved. We investigated the possibility of using an inducible system to increase the proportion of constitutively active wild-type FLAG-hAT(1)Rs by overexpressing G(q)alpha. Following doxycycline treatment, we observed no significant change in the apparent binding affinity or potency (coupling efficiency) of angiotensin II, though significant increases in the intrinsic activity of several partial agonists were observed, indicative of constitutive activity. DUP753 (10 microM), a suggested inverse agonist, did not inhibit the enhanced level of basal (agonist-independent) activity. The data suggest that the resting equilibrium of hAT(1) receptors between the inactive (R) and active (R*) forms is predominantly weighted towards the inactive conformation.
Scragg
Jason L
JL
CRISTAL (Cardiovascular Research Institute at Leeds), School of Medicine, University of Leeds, Leeds LS2 9JT, UK. J.Scragg@leeds.ac.uk
Warburton
Philip
P
Ball
Stephen G
SG
Balmforth
Anthony J
AJ
D000804:Angiotensin II / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004318:Doxycycline / Q000494:pharmacology; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007668:Kidney / Q000187:drug effects / Q000378:metabolism*; D044140:Receptor, Angiotensin, Type 1 / Q000819:agonists* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D044140:Receptor, Angiotensin, Type 1; D011994:Recombinant Proteins; D000804:Angiotensin II; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D004318:Doxycycline; D002118:Calcium