PT  - JOURNAL ARTICLE
AU  - Joseph B. Thompson
AU  - Susan M. Wade
AU  - Jeffrey K. Harrison
AU  - Mina N. Salafranca
AU  - Richard R. Neubig
TI  - Cotransfection of Second and Third Intracellular Loop Fragments Inhibit Angiotensin AT1a Receptor Activation of Phospholipase C in HEK-293 Cells
DP  - 1998 Apr 01
TA  - Journal of Pharmacology and Experimental Therapeutics
PG  - 216--222
VI  - 285
IP  - 1
4099  - http://jpet.aspetjournals.org/content/285/1/216.short
4100  - http://jpet.aspetjournals.org/content/285/1/216.full
SO  - J Pharmacol Exp Ther1998 Apr 01; 285
AB  - Peptides from the intracellular regions of G protein-coupled receptors are useful probes of receptor-G protein coupling mechanisms. As a first step toward the genetic delivery of such “G protein inhibitors,” we describe inhibition of angiotensin II (AII) receptor responses by expressed fragments of the second and third intracellular loops of the AT1a receptor (AT1a/i2 and AT1a/i3). Transient transfection of human embryonic kidney 293 cells with DNA encoding the rat AT1a receptor resulted in AII-dependent increases of inositol phosphates (maximum 4.5-fold). Cotransfection of AT1a/i2 and AT1a/i3 fragments raised the EC50 for AII 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 was additive, and transfection of an alpha-1b adrenergic receptor third intracellular loop (α1b/i3) fragments also increased the EC50 for AII. Neither AT1a/i1 nor C-terminus (AT1a/Ct) constructs had significant effects on angiotensin responses. These data confirm a role for the second and third intracellular loops in angiotensin receptor responses and show the potential of this approach to blocking multiple phospholipase C-linked receptors. The American Society for Pharmacology and Experimental Therapeutics