COTRANSFECTION OF 2ND-INTRACELLULAR AND 3RD-INTRACELLULAR LOOP FRAGMENTS INHIBIT ANGIOTENSIN AT1A RECEPTOR ACTIVATION OF PHOSPHOLIPASE-C INHEK-293 CELLS

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.