Electrostatic and Hydrophobic Forces Tether the Proximal Region of the Angiotensin II Receptor (AT1A) Carboxyl Terminus to Anionic Lipids

The carboxyl terminus of the type-1 angiotensin II receptor (AT1A) is a focal point for receptor activation and deactivation. Synthetic peptides corresponding to the membrane-proximal, first 20 amino acids of the carboxyl terminus adopt an α-helical conformation in organic solvents, suggesting that the secondary structure of this region may be sensitive to hydrophobic environments. Using surface plasmon resonance, immobilized lipid chromatography, and circular dichroism, we examined whether this positively charged, amphipathic α-helical region of the AT1A receptor can interact with lipid components in the cell membrane and thereby modulate local receptor attachment and structure. A synthetic peptide corresponding to the proximal region of the AT1A receptor carboxyl terminus (Leu305 to Lys325) was shown by surface plasmon resonance to bind with high affinity to the negatively charged lipid, dimyristoyl l-α-phosphatidyl-dl-glycerol (DMPG), but poorly to the zwitterionic lipid, dimyristoyl l-α-phosphatidylcholine (DMPC). In contrast, a peptide analogue possessing substitutions at four lysine residues (corresponding to Lys307,308,310,311) displayed poor association with either lipid, indicating a crucial anionic component to the interaction. Circular dichroism analysis revealed that both the wild-type and substituted peptides possessed α-helical propensity in methanol and trifluoroethanol, while the wild-type peptide also adopted partially inserted helical structure in DMPG and DMPC liposomes. In contrast, the substituted peptide exhibited spectra that suggested the presence of β-sheet and α-helical structure in both liposomes. Immobilized lipid chromatography was used to characterize the hydrophobic component of the membrane interaction, and the results demonstrated that hydrophobic and electrostatic interactions mediated the binding of the wild-type peptide but that the substituted peptide bound to the model membranes mainly via hydrophobic forces. We propose that, in intact AT1A receptors, the proximal carboxyl terminus associates with the cytoplasmic face of the cell membrane via a high-affinity, anionic phospholipid-specific tethering that serves to increase the amphipathic helicity of this region. Such associations may be important for receptor function and common for G protein-coupled receptors

A single beta-amino acid substitution to angiotensin II confers AT(2) receptor selectivity and vascular function

Novel AT2R ligands were designed by substituting individual β-amino acid in the sequence of the native ligand angiotensin II (Ang II). Relative ATR selectivity and functional vascular assays (in vitro AT2R-mediated vasorelaxation and in vivo vasodepressor action) were determined. In competition binding experiments using either AT1R- or AT2R- transfected HEK-293 cells, only β-Asp-Ang II and Ang II fully displaced [I]-Ang II from AT1R. In contrast, β-substitutions at each position of Ang II exhibited AT2R affinity, with β-Tyr-Ang II and β-Ile-Ang II exhibiting 1000-fold AT2R selectivity. In mouse aortic rings, β-Tyr-Ang II and β-Ile-Ang II evoked vasorelaxation that was sensitive to blockade by the AT2R antagonist PD123319 and the nitric oxide synthase inhibitor L-NAME. When tested with a low level of AT1R blockade, β-Ile-Ang II (15 pmol/kg per minute IV for 4 hours) reduced blood pressure (BP) in conscious spontaneously hypertensive rats (β-Ile-Ang II plus candesartan, -24±4 mm Hg) to a greater extent than candesartan alone (-11±3 mm Hg, n=7,

The β-amyloid protein of Alzheimer's disease increases neuronal CRMP-2 phosphorylation by a Rho-GTP mechanism

Neuritic abnormalities are a major hallmark of Alzheimer's disease (AD) pathology. Accumulation of β-amyloid protein (Aβ) in the brain causes changes in neuritic processes in individuals with this disease. In this study, we show that Aβ decreases neurite outgrowth from SH-SY5Y human neuroblastoma cells. To explore molecular pathways by which Aβ alters neurite outgrowth, we examined the activation and localization of RhoA and Rac1 which regulate the level and phosphorylation of the collapsin response mediator protein-2 (CRMP-2). Aβ increased the levels of the GTP-bound (active) form of RhoA in SH-SY5Y cells. This increase in GTP-RhoA correlated with an increase in an alternatively spliced form of CRMP-2 (CRMP-2A) and its threonine phosphorylated form. Both a constitutively active form of Rac1 (CA-Rac1) and the Rho kinase inhibitor, Y27632, decreased levels of the CRMP-2A variant and decreased threonine phosphorylation caused by Aβ stimulation. The amount of tubulin bound to CRMP-2 was decreased in the presence of Aβ but Y27632 increased the levels of tubulin bound to CRMP-2. Increased levels of both RhoA and CRMP-2 were found in neurons surrounding amyloid plaques in the cerebral cortex of the APP(Swe) Tg2576 mice. We found that there was an increase in threonine phosphorylation of CRMP-2 in Tg2576 mice and the increase correlated with a decrease in the ability of CRMP-2 to bind tubulin. The results suggest that Aβ-induced neurite outgrowth inhibition may be initiated through a mechanism in which Aβ causes an increase in Rho GTPase activity which, in turn, phosphorylates CRMP-2 to interfere with tubulin assembly in neurites