Cotransfection of Second and Third Intracellular Loop Fragments Inhibit Angiotensin AT1a Receptor Activation of Phospholipase C in HEK-293 Cells1
- Joseph B. Thompson1,
- Susan M. Wade1,
- Jeffrey K. Harrison3,
- Mina N. Salafranca3 and
- Richard R. Neubig1,2
- Departments of 1Pharmacology (J.B.T., S.M.W., R.R.N.) and 2Internal Medicine/Hypertension (R.R.N.), The University of Michigan, Ann Arbor, Michigan and the 3Department of Pharmacology and Therapeutics (M.N.S., J.K.H.), University of Florida, Gainesville, Florida
Abstract
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.
Footnotes
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Send reprint requests to: Richard Neubig, M.D., Ph.D., Department of Pharmacology, 1301 MSRB III, 1150 W. Med. Ctr. Dr., Ann Arbor, MI 48109-0632.
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↵1 Support for this work was provided by NIH HL46417 (RRN), T32GM07863 (JBT) and a Grant-in-Aid, AHA-Florida (JKH).
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↵2 We did not determine theKd for studies with coexpression of the AT/i1 construct. The binding data at single-ligand concentrations did not show any significant differences from control.
- Abbreviations:
- AT1aR
- angiotensin 1a receptor
- α1b-AR
- alpha1b adrenergic receptor
- GPCR
- G protein-coupled receptor
- HEK-293
- human embryonic kidney 293 cells
- i1
- first intracellular loop
- i2
- second intracellular loop
- i3
- third intracellular loop
- C-term
- C-terminal tail
- IPx
- inositol phosphates
- pAT1aR
- plasmid encoding rat AT1aR
- PCR
- polymerase chain reaction
- PLC
- phospholipase C
- AII
- angiotensin II
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- Received June 17, 1997.
- Accepted December 19, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
