Reduction of Blood Pressure by AT₁ Receptor Decoy Peptides

INTRODUCTION

Angiotensin II (AngII), acting predominantly via its AT₁ receptor (AT₁R), plays important roles in the regulation of blood pressure (BP) and intravascular volume. AT₁'s action is often targeted in the treatment of hypertension and other disorders.^1-12 We previously identified the binding of the chaperone protein gamma-aminobutyric acid receptor–associated protein (GABARAP) to a sequence on the carboxy-terminus of the AT₁ receptor (AT₁R) and showed that this binding enhances AT₁R trafficking to the cell surface as well as angiotensin signaling.^13,14 To determine the effect of inhibiting receptor/chaperone interaction in vivo, we treated sodium-depleted mice with decoy peptides consisting of either a fusion of the cell-penetrating peptide (CPP) penetratin and the GABARAP/AT₁R binding sequence of AT₁R or a fusion of penetratin and a mutated AT₁R sequence. We used telemetry to measure BP.

RESULTS

CPP-1 decreased 24-hour average systolic BP from 129.8 ± 4.7 mmHg to 125.0 ± 6.0 mmHg (mean ± standard deviation). Diastolic BP fell from 99.0 ± 7.1 mmHg to 95.0 ± 9.2 mmHg (n=5). CPP-2 raised systolic BP from 128.7 ± 1.3 mmHg to 131.7 ± 2.9 mmHg and diastolic BP from 93.9 ± 4.5 mmHg to 95.9 ± 4.2 mmHg (n=5). The decreases in both systolic and diastolic BP after administration of the active peptide were statistically significant compared to changes after administration of the control peptide (P<0.05, two-tailed Wilcoxon rank-sum test).

DISCUSSION

AngII is the major effector protein of the renin-angiotensin system. It acts on vascular smooth muscle cells to induce vasoconstriction and on adrenal cortical cells to stimulate aldosterone secretion. Both of these actions increase BP. The peptide also can bind to receptors in the brain and affect the neural control of BP. Therefore, AngII is an important factor in the maintenance of normal BP as well as in the pathogenesis of hypertension.^1-6 Indeed, drugs designed to reduce the production of AngII (direct renin inhibitors and converting enzyme inhibitors) or to block its action at AT₁R—AngII's predominant cellular receptor—(angiotensin-receptor blockers) are widely used antihypertensive agents. Also, AngII is directly involved in the production of atherosclerosis, cardiac hypertrophy, congestive heart failure, and diabetic nephropathy and other renal diseases. As noted, the majority of AngII-mediated physiological actions occur through binding to the AT₁R, a 7-membrane–spanning G protein–coupled receptor.^1-6

AT₁Rs are not static on cell surfaces but can be internalized after AngII binding and either recycled to the cell surface or trafficked to other intracellular compartments. Chaperone proteins can modulate the trafficking of AT₁R to and from the cell surface. One chaperone protein is the angiotensin receptor–associated protein (ATRAP) that reduces trafficking of the receptor to the cell surface. The upregulation of ATRAP by physiological means can lessen the ability of hypertension to damage the kidneys of Dahl salt-sensitive rats, presumably by reducing AT₁R sites on renal cells and thereby limiting the harmful action of AngII on these cells.^7,8 A second chaperone protein is ARAP1 that, unlike ATRAP, facilitates trafficking of AT₁R to the cell surface.^9-12

We discovered that another chaperone protein, GABARAP, like ARAP1, binds to the carboxy-terminus of AT₁R and promotes the trafficking of the receptor to the cell surface.^13,14 GABARAP has been known to affect gamma-amino butyric acid receptor trafficking and clustering in brain neurons but previously was not known to interact with AT₁R. In cell cultures, cotransfection of PC-12 cells (a pheochromocytoma cell line) with a fluorescent AT₁R fusion protein and GABARAP increased AT₁R cell surface expression by 6-fold. GABARAP overexpression in CHO-K1 cells that also expressed AT₁R increased cell surface AngII binding more than 3-fold and increased AngII-driven signaling and proliferation as well. Knockdown of GABARAP with small interfering RNAs reduced AT₁R surface protein and binding.¹³

We next identified GABARAP/AT₁R interacting sites on each protein and a target sequence in the carboxy-terminus of AT₁R (GKKFKKYFLQL). We transfected mammalian cells with AT₁R and GABARAP and then, in a proof-of-concept experiment, treated the cells externally with cell-penetrating decoy peptides.^13,15 These peptides consisted of fusions of penetratin with GKKFKKYFLQL (AT₁R) or GKKFEEAFLQL (mutated AT₁R). The active peptide is designated CPP-1, and the control peptide is CPP-2. Deconvolution microscopy and immunoblot studies showed that the active decoy CPPs blocked GABARAP-induced AT₁R accumulation at the cell surface and blocked AngII-induced stimulation of phospho–extracellular signal-regulated protein kinases 1 and 2 by about 5-fold. CPPs fused to mutant AT₁R sequences had no effect.¹⁵

Research has shown AngII to be necessary to maintain normal BP in sodium-depleted, but not sodium-replete, humans.¹⁶ We designed the present study to demonstrate the in vivo BP-lowering action of inhibitors of the GABARAP/AT₁R interaction by treating sodium-depleted mice with GABARAP/AT₁R decoy peptides.

This study demonstrates the in vivo relevance of the GABARAP/AT₁R interaction for BP and demonstrates the efficacy of decoy CPPs in lowering BP. This finding suggests that small molecule inhibitors could be developed to block the GABARAP/AT₁R interaction site and lower BP, as well as potentially reduce other harmful effects of AngII by actions at arterial smooth muscle or other sites.^1,4-6 Although the pressure reduction achieved in this study was modest, it was comparable to average pressure decreases obtained through the use of angiotensin-converting enzyme inhibitors in several large clinical trials.¹⁷ Moreover, the decrease in BP reported here occurred in normotensive animals. Greater decreases would be expected in hypertensive animals, particularly animals with high renin hypertension. Like angiotensin-converting enzyme inhibitors and angiotensin receptor blockers, these peptides or their small molecule analogues could find wide application in treating congestive heart failure, diabetic renal disease, and other disorders.

Because large proteins such as the decoy peptides described here do not cross the blood-brain barrier, the effects we observed are likely related to AT₁R in the cardiovascular system, suggesting that a different spectrum of activity could result from the delivery of these decoy peptides or their small molecule analogues into the central nervous system.

Additionally, the active decoy peptide described here lowers cell surface receptor number and, thereby, not only reduces AngII signaling but also reduces AT₁R-mediated AngII internalization and therefore any effects attendant upon that internalization.^1-4,18 Similarly, to the extent that constitutive AT₁R activity requires trafficking to the cell membrane, interruption of the GABARAP/AT₁R interaction by decoy peptides would be expected to blunt that activity.¹⁹

CONCLUSION

To our knowledge, this is the first report of a study showing that the inhibition of a chaperone protein binding to AT₁R can lower BP in vivo. This observation potentially has considerable therapeutic implications.