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

Abstract

Hemorphins are known for their role in the control of blood pressure. Recently, we revealed the positive modulation of the angiotensin II (AngII) type 1 receptor (AT1R) by LVV-hemorphin-7 (LVV-H7) in human embryonic kidney (HEK293) cells. Here, we examined the molecular binding behavior of LVV-H7 on AT1R and its effect on AngII binding using a nanoluciferase-based bioluminescence resonance energy transfer (NanoBRET) assay in HEK293FT cells, as well as molecular docking and molecular dynamics (MD) studies. Saturation and real-time kinetics supported the positive effect of LVV-H7 on the binding of AngII. While the competitive antagonist olmesartan competed with AngII binding, LVV-H7 slightly, but significantly, decreased AngII’s kD by 2.6 fold with no effect on its Bmax. Molecular docking and MD simulations indicated that the binding of LVV-H7 in the intracellular region of AT1R allosterically potentiates AngII binding. LVV-H7 targets residues on intracellular loops 2 and 3 of AT1R, which are known binding sites of allosteric modulators in other GPCRs. Our data demonstrate the allosteric effect of LVV-H7 on AngII binding, which is consistent with the positive modulation of AT1R activity and signaling previously reported. This further supports the pharmacological targeting of AT1R by hemorphins, with implications in vascular and renal physiology.

Investigation of Receptor Heteromers Using NanoBRET Ligand Binding

“…Several studies have investigated this heteromer in a variety of systems, and reported evidence for it being a constitutive heteromer that displays modulated G protein signaling and β-arrestin recruitment [9,24,25]. To investigate this complex we first looked at binding of a BODIPY-630/650 tagged angiotensin II ligand (BODIPY-AngII) to the N-terminally Nluc-tagged AT 1 receptor (Nluc-AT 1 ; Figure 2a) using the NanoBRET ligand binding assay [18][19][20][21]. We were able to demonstrate saturable binding of BODIPY-AngII to the AT 1 receptor, which was substantially reduced by treatment with a high concentration of the AT 1 antagonist olmesartan (Figure 2b), enabling generation of a BODIPY-AngII specific binding curve (Figure 2c).…”

“…Recently, we have developed the NanoBRET ligand binding assay [18][19][20][21][22][23], in which we have successfully monitored binding of fluorescent ligands to Nanoluciferase (Nluc)tagged GPCRs. In the present study, we have demonstrated the Receptor-HIT assay utilizing NanoBRET ligand binding to investigate two GPCR heteromers: between the angiotensin II type 1 (AT 1 ) receptor and the β 2 adrenergic receptor (AT 1 -β 2 AR heteromer), as well as between the AT 1 and angiotensin II type 2 receptor (AT 1 -AT 2 heteromer).…”

Hemorphins—From Discovery to Functions and Pharmacology

“…The expressed receptor was the GPCR-type complex, involving recruitment of beta-arrestin upon stimulation, followed by the ligand binding, and an interesting conclusion was drawn concerning the role of hemorphins in analgesia. This group also examined the binding behavior of LVV-H7 to AT1R and its effect on angiotensin II binding using nanoluciferasebased bioluminescence resonance energy transfer (NanoBRET) in HEK293FT cells [54].…”

“…It was shown that camel LVV-H7 produced stronger interactions with all studied proteins (MOR, ACE and IRAP) than non-camel LVV-H7. A year later, Ali et al presented application of molecular docking to investigate binding of LVV-H7 to AT1R receptor [54]. It was reported that the performed interaction indicates that docking mechanism of LVV-H7 to angiotensin II type 1 receptor (AT1R) allosterically potentiates angiotensin II binding.…”

“…Hemorphins are also recognized for their role in the blood pressure control, as the positive modulation of the angiotensin II (AngII) type 1 receptor (AT1R) by LVV-H7 was described by Ali A et al [53]. Other studies were performed to study the role of LVV-H7 in vascular and renal systems [46,54]. Consequently, LVV-H7, or its analogs, may be used as a therapeutic reagent for blood pressure control or as a biomarker of a vascular disease.…”