Cell-Penetrating Peptides Corresponding to the Angiotensin II Type 1 Receptor Reduce Receptor Accumulation and Cell Surface Expression and Signaling

Abstract

Our previous published studies have established the γ-aminobutyric acid (GABA) receptor-associated protein (GABARAP) as a trafficking protein for the angiotensin II type 1A receptor (AT(1)R). GABARAP overexpression increases both AT(1)R protein accumulation and translocation to the plasma membrane. The present study examined the inhibitory effects of decoy peptides on receptor expression and plasma membrane accumulation. The decoy peptides correspond to the AT(1)R cytoplasmic domain located immediately proximal to the 7th transmembrane domain, a region implicated in GABARAP binding. This competitive binding study was designed as a first step toward evaluating the GABARAP:AT(1)R binding interface as a target for reducing AT(1)R trafficking to the plasma membrane. AT(1)R and GABARAP plasmids were transfected into mammalian cell lines simultaneously with cell-penetrating peptides (CPPs). CPP-1 and CPP-2 consist of the penetratin (pANT(43-58)) CPP with downstream fusions of GKKFKKYFLQL (AT(1)R) and GKKFEEAFLQL (AT(1)R-mutant) amino acids, respectively. CPP-3 consists of the HIV TAT(48-60) CPP with GKKFKKYFLQL (AT(1)R) fused downstream. Western blotting, signal transduction studies, and 3D deconvolution microscopy experiments were employed. Immunoblot analyses and live cell deconvolution microscopy demonstrated that inhibitory (but not control) peptides completely blocked GABARAP-induced intracellular AT(1)R accumulation and cell surface accumulation. GABARAP also stimulated angiotensin II-mediated phospho-ERK1/2 induction by ~ fivefold. This activation was, similarly, quantitatively blocked by the inhibitory peptides. Cell-penetrating decoy peptides which were designed to block the AT(1)R:GABARAP interaction, effectively reduced AT(1)R intracellular accumulation and cell-surface trafficking and signaling. The binding interaction site between AT(1)R and GABARAP represents a potential therapeutic target.