Interplay Between Angiotensin II Type 1 Receptor and Thrombin Receptor Revealed by Bioluminescence Resonance Energy Transfer Assay

Abstract

The key hormone of the renin-angiotensin system (RAS), angiotensin II (AngII), and thrombin are known to play major roles in the vascular system and its related disorders. Previous studies reported connections between AngII and thrombin in both physiological and pathophysiological models. However, the molecular mechanisms controlling such interplay at the level of their receptors belonging to the family of G protein-coupled receptors (GPCRs) are not fully understood. In this study, we investigated the functional interaction between the AngII type 1 receptor (AT1R) and the thrombin receptor [or protease-activated receptor 1 (PAR1)] in human embryonic kidney 293 (HEK293) cells. For this, we used various bioluminescence resonance energy transfer (BRET) proximitybased assays to profile the coupling to the heterotrimeric Gaq protein, b-arrestin recruitment, and receptor internalization and trafficking in intact cells. The overall doseresponse and real-time kinetic BRET data demonstrated the specific molecular proximity between AT1R and PAR1 resulting in their functional interaction. This was characterized by thrombin inducing BRET increase within AT1R/Gaq and AT1R/b-arrestin pairs and synergistic effects observed upon the concomitant activation of both receptors suggesting a positive allosteric interaction. The BRET data corroborated with the data on the downstream Gaq/inositol phosphate pathway. Moreover, the selective pharmacological blockade of the receptors revealed the implication of both AT1R and PAR1 protomers in such a synergistic interaction and the possible transactivation of AT1R by PAR1. Interestingly, the positive action of PAR1 on AT1R activation was contrasted with its apparent inhibition of AT1R internalization and its endosomal trafficking. Finally, BRET saturation and co-immunoprecipitation assays supported the physical AT1-PAR1 interaction in HEK293 cells. Our study reveals for the first time the functional interaction between AT1R and PAR1 in vitro characterized by a transactivation and positive allosteric modulation of AT1R and inhibition of its desensitization and internalization. This finding may constitute the molecular basis of the well-known interplay between RAS and thrombin. Thus, our data should lead to revising some findings on the implication of

Synergistic activation of thrombin and angiotensin II receptors revealed by bioluminescence resonance energy transfer

“…Such trans‐activation required to a certain extent the activate conformation of PAR1 since it was partially blocked by PAR1 antagonist. More interestingly, we also found that the dual activation of AT1R and PAR1 led to synergistic effects regarding AT1R‐Gαq coupling and AT1R‐β‐arrestin association that were drastically inhibited by both AT1R and PAR1 selective antagonists [4]. This suggests a positive allosteric interaction between the two protomers both required to be active within the AT1R‐PAR1 heterodimer.…”

“…The major advances in the field of GPCR heterodimerization were possible thank to the development and application of various techniques such as the bioluminescence and fluorescence resonance energy transfer (BRET and FRET) and their derived assays allowing the study of GPCR pharmacology and signaling from multiple angles and prospects [3]. In this context, we recently reported an interesting functional interplay between the angiotensin II (AngII) type 1 receptor (AT1R) and the thrombin or protease‐activated receptor 1 (PAR1) in the human embryonic kidney (HEK293) cells using BRET technology [4]. This may be the molecular basis of the previous in vitro and in vivo studies reporting the interaction between the renin‐angiotensin system (RAS) and the protease thrombin in the vascular and renal systems [5–9].…”

“…In our recent study, we demonstrated the physical and functional interaction between AT1R and PAR1, which was characterized by an interesting pharmacological fingerprint. Indeed, we showed that thrombin and PAR1 trans‐activated AT1R in dose‐ and time‐dependent manners even in the absence of stimulation with AngII [4]. This occurred with respect to AT1R‐Gαq coupling and AT1R‐β‐arrestin association in HEK293 cells.…”

“…This suggests a positive allosteric interaction between the two protomers both required to be active within the AT1R‐PAR1 heterodimer. Furthermore, PAR1 and its activation with thrombin was found to inhibit AngII‐promoted AT1R internalization and endosomal trafficking [4]. We speculated on the possibility that AT1R and PAR1 adopt specific conformations within AT1R‐PAR1 complexes leading to potentiated Gαq activation and β‐arrestin recruitment, which by contrast, inhibited AT1R internalization.…”

Profiling novel pharmacology of receptor complexes using Receptor-HIT

“…Receptor-HIT has most commonly been used to investigate the pharmacology of receptor heteromers composed of two different GPCRs (termed GPCR-HIT) [10,12,[17][18][19][20][21][22][23][24]. GPCRs are the largest family of membrane receptors and are also the largest family of drug targets, with over 30% of currently approved drugs acting via GPCRs [25][26][27].…”