Angiotensin II through its AT1 receptor participates in the etiology of a number of diseases, such as hypertension and fibrotic diseases. Although its overall actions can be regulated by preventing its formation with ACE inhibitors or blocking its overall action with AT1 receptor blockers, the mechanisms of it actions are essentially unknown. A very proficient way to study the signaling by the AT1 receptor is through its signal motif manipulation by mutagenesis. This approach provides information as to the actions and interactions of the motifs but does not provide an opportunity to regulate the action of the wild type receptor. This communication is an initial attempt to make use of cell penetrating peptides to both delineate and regulate the action(s) of the AT1 receptor.
In previous studies we demonstrated that AngII, through the AT1 receptor, increases Akt phosphorylation in AT1 cDNA tranfected HEK-293 cells while bradykinin decreases it in bradykinin B2 receptor cDNA transfected cells (10
). In HEK-293 cells transfected with a mutant AT1 receptor containing the IC2 of the BKB2R receptor AngII decreased pAkt (10
) thus assigning regulation of Akt phosphorylation. In contrast, these receptor manipulations at the level of the IC2 of the AT1R had no effect on the activation of ERK by the mutant receptor as compared to the WT AT1 receptor. Thus the IC2 of the AT1R is an important signaling motif with respect to both G-protein associated signaling as exemplified by PI turnover and Akt activation, whereas IC2 seems not involved in ERK activation. It has been shown that AngII activates ERK by a Gαq-independent action (15
). Studies using carboxyl terminal-truncated AT1 receptors indicate that the amino acid sequence between 312 and 337 is required for the activation of ERK. The YIPP motif within this sequence appears to be the important motif in this action (16
). Mutations in the highly conserved D125
sequence of the AT1R result in a receptor that is not coupled to Gαq, but still activates ERK (17
In the past few years there has been a progressive development of membrane-penetrating peptides which has allowed for the delivery of otherwise impermeable bioactive molecules across the plasma membrane. Numerous studies using 10–100μM CPP concentrations have demonstrated that once accumulated in specific compartments (i.e., the cytosol, nucleus and mitochondria) the specific cargo can regulate targeted cellular activities through protein-protein interactions within the intact cells (18
). The results obtained here utilizing the AT1-IC2/HIV TAT cell penetrating peptide confirmed our previous observations and illustrated that the introduction of competing peptide into the cytosol mimicking a receptor motif (IC2) can indeed alter receptor signaling ability. In predominant number of reports 10–100 μM CPP concentrations have been used to demonstrate the effect of these peptides. We chose the lowest concentration of this span in order to limit as much as possible any non specific effects of TAT. TAT was shown to rapidly transduce large cargos such as CRE recombinase into primary cells (19
). Thus the size of the cargo does not limit the cell penetrating ability of TAT. As shown by the 5-FAM/FACS experiment carried out as part of these studies the TAT peptide clearly crossed into the cytosol of both the HEK-293 and the pulmonary smooth muscle cells. The AT1 IC2 peptide effectively inhibited Gαq related signaling such as PI turnover and Ca2+
influx in HEK-293 cells. In addition, it limited the AngII dependent activation of Akt/PKB and NFκB, but had no effect on ERK activation in HPASMCs. We used the TAT-linked second intracellular loop of the bradykinin B2 receptor as the control peptide. This peptide had no effect on AngII induced signaling actions. Thompson et al showed that cotransfecting second and third loops inhibit AT1 receptor activation of PLC in HEK-293 cells (20
). Our peptide approach fits their results. Vazquez et al reported that the synthesized third intracellular loop of the AT1 receptor linked to HIV-TAT was able to be transduced into the hypothalamus and brainstem neurons and increased neuronal firing rate, an effect similar to that observed with angiotensin II stimulation of the neuronal AT1R (21
). A more recent study demonstrated that a peptide based on the C-terminus of the AT1 receptor with native cell-penetrating sequences elicits G-protein mediated blood vessel contraction (22
), though our IC2 peptide seems evoking an inhibitory effect. Therefore, the activating or inhibiting effects of the intracellular motifs in GPCRs are subtle and complicated. Further structural studies such as molecular modeling or NMR could elucidate the accurate mechanisms of those effects including possible “off target” effects of the peptides.
In summary, our results with cell/tissue penetrating peptides are very encouraging, and are leading us to anticipate that these peptides will contribute to the control of receptor initiated signaling and blood vessel function.