Jb. Thompson et al., COTRANSFECTION OF 2ND-INTRACELLULAR AND 3RD-INTRACELLULAR LOOP FRAGMENTS INHIBIT ANGIOTENSIN AT1A RECEPTOR ACTIVATION OF PHOSPHOLIPASE-C INHEK-293 CELLS, The Journal of pharmacology and experimental therapeutics, 285(1), 1998, pp. 216-222
Peptides from the intracellular regions of G protein-coupled receptors
are useful probes of receptor-G protein coupling mechanisms. As a fir
st step toward the genetic delivery of such ''G protein inhibitors,''
we describe inhibition of angiotensin II (All) receptor responses by e
xpressed fragments of the second and third intracellular loops of the
AT1a receptor (AT1a/i2 and AT1a/i3). Transient transfection of human e
mbryonic kidney 293 cells with DNA encoding the rat AT1a receptor resu
lted in All-dependent increases of inositol phosphates (maximum 4.5-fo
ld). Cotransfection of AT1a/i2 and AT1a/i3 fragments raised the EC50 f
or All stimulation of phospholipase C activity 5-fold (from 0.18 nM to
0.99 nM, n = 12, P < .001) and 3-fold (from 0.38 nM to 1.2 nM, n = 8,
P < .002), respectively. The combined effect of AT1a/i2 and AT1a/3 wa
s additive, and transfection of an alpha-lb adrenergic receptor third
intracellular loop (alpha 1b/i3) fragments also increased the EC50 for
All. Neither AT1a/i1 nor C-terminus (AT1a/C-t) constructs had signifi
cant effects on angiotensin responses. These data confirm a role for t
he second and third intracellular loops in angiotensin receptor respon
ses and show the potential of this approach to blocking multiple phosp
holipase C-linked receptors.