Positive Modulation of Angiotensin II Type 1 Receptor–Mediated Signaling by LVV–Hemorphin-7

Abstract

Hemorphins are hemoglobin β-chain–derived peptides initially known for their analgesic effects via binding to the opioid receptors belonging to the family of G protein–coupled receptor (GPCR), as well as their physiological action on blood pressure. However, their molecular mechanisms in the regulation of blood pressure are not fully understood. Studies have reported an antihypertensive action via the inhibition of the angiotensin-converting enzyme, a key enzyme in the renin–angiotensin system. In this study, we hypothesized that hemorphins may also target angiotensin II (AngII) type 1 receptor (AT1R) as a key GPCR in the renin–angiotensin system. To investigate this, we examined the effects of LVV–hemorphin-7 on AT1R transiently expressed in human embryonic kidney (HEK293) cells using bioluminescence resonance energy transfer (BRET) technology for the assessment of AT1R/Gαq coupling and β-arrestin 2 recruitment. Interestingly, while LVV–hemorphin-7 alone had no significant effect on BRET signals between AT1R and Gαq or β-arrestin 2, it nicely potentiated AngII-induced BRET signals and significantly increased AngII potency. The BRET data were also correlated with AT1R downstream signaling with LVV–hemorphin-7 potentiating the canonical AngII-mediated Gq-dependent inositol phosphate pathway as well as the activation of the extracellular signal–regulated kinases (ERK1/2). Both AngII and LVV–hemorphin-7–mediated responses were fully abolished by AT1R antagonist demonstrating the targeting of the active conformation of AT1R. Our data report for the first time the targeting and the positive modulation of AT1R signaling by hemorphins, which may explain their role in the physiology and pathophysiology of both vascular and renal systems. This finding further consolidates the pharmacological targeting of GPCRs by hemorphins as previously shown for the opioid receptors in analgesia opening a new era for investigating the role of hemorphins in physiology and pathophysiology via the targeting of GPCR pharmacology and signaling.

Camel Hemorphins Exhibit a More Potent Angiotensin-I Converting Enzyme Inhibitory Activity than Other Mammalian Hemorphins: An In Silico and In Vitro Study

“…A number of studies have highlighted their therapeutic potential [10,[17][18][19]47,48]. We had previously reported the molecular binding behavior of camel and non-camel LVV-hemorphin-7 on multiple targets [20,25]. Here, we report the binding and ACE inhibition potential of non-camel hemorphins (LVVYPWTQRF and YPWTQRF) and camel hemorphins (LVVYPWTRRF and YPWTRRF) using computational approaches and an in vitro ACE inhibition assay.…”

“…It is also the most stable and has the highest hydrophobicity when compared to other hemorphins [15,16]. Numerous studies have reported various biological activities of hemorphins, such as effects on spatial learning, analgesia, and hypertension [10,[17][18][19][20]. Hemorphins have been shown to have beneficial effects in the control of blood pressure.…”

Insights into the Interaction of LVV-Hemorphin-7 with Angiotensin II Type 1 Receptor

“…We previously showed a positive allosteric modulation of AT1R activity and signaling by LVV-H7 in HEK293 cells [ 14 ]. In this study, we examined the effect of LVV-H7 on the binding of AngII on AT1R using NanoBRET technology in real-time and intact cells as previously reported [ 15 ].…”

“…Inhibition of ACE leads to reduced angiotensin II (AngII) levels, which are associated with the well-known anti-hypertensive properties of hemorphins [ 6 , 12 , 13 ]. Recently, we described for the first time the AngII type 1 receptor (AT1R) as a second GPCR to be pharmacologically targeted by the decapeptide LVV-hemorphin-7 (LVV-H7) in vitro with a positive modulation of its activity and downstream signaling pathways in HEK293 cells [ 14 ]. These effects were observed on the canonical Gαq/inositol phosphate pathway of AT1R, as well as on the phosphorylation of the extracellular signal-regulated-kinases (ERK1/2) [ 14 ].…”

“…Recently, we described for the first time the AngII type 1 receptor (AT1R) as a second GPCR to be pharmacologically targeted by the decapeptide LVV-hemorphin-7 (LVV-H7) in vitro with a positive modulation of its activity and downstream signaling pathways in HEK293 cells [ 14 ]. These effects were observed on the canonical Gαq/inositol phosphate pathway of AT1R, as well as on the phosphorylation of the extracellular signal-regulated-kinases (ERK1/2) [ 14 ]. The positive modulation of AT1R implies a hypertensive effect and this was surprising and not consistent with the anti-hypertensive properties of hemorphins through the inhibition of ACE.…”