Evidence for Heterodimerization and Functional Interaction of the Angiotensin Type 2 Receptor and the Receptor MAS

Abstract

The angiotensin type 2 receptor (AT2R) and the receptor MAS are receptors of the protective arm of the renin-angiotensin system. They mediate strikingly similar actions. Moreover, in various studies, AT2R antagonists blocked the effects of MAS agonists and vice versa. Such cross-inhibition may indicate heterodimerization of these receptors. Therefore, this study investigated the molecular and functional interplay between MAS and the AT2R. Molecular interactions were assessed by fluorescence resonance energy transfer and by cross correlation spectroscopy in human embryonic kidney-293 cells transfected with vectors encoding fluorophore-tagged MAS or AT2R. Functional interaction of AT2R and MAS was studied in astrocytes with CX3C chemokine receptor-1 messenger RNA expression as readout. Coexpression of fluorophore-tagged AT2R and MAS resulted in a fluorescence resonance energy transfer efficiency of 10.8 ± 0.8%, indicating that AT2R and MAS are capable to form heterodimers. Heterodimerization was verified by competition experiments using untagged AT2R and MAS. Specificity of dimerization of AT2R and MAS was supported by lack of dimerization with the transient receptor potential cation channel, subfamily C-member 6. Dimerization of the AT2R was abolished when it was mutated at cysteine residue 35. AT2R and MAS stimulation with the respective agonists, Compound 21 or angiotensin-(1-7), significantly induced CX3C chemokine receptor-1 messenger RNA expression. Effects of each agonist were blocked by an AT2R antagonist (PD123319) and also by a MAS antagonist (A-779). Knockout of a single of these receptors made astrocytes unresponsive for both agonists. Our results suggest that MAS and the AT2R form heterodimers and that-at least in astrocytes-both receptors functionally depend on each other.

Angiotensin II Type 2 Receptor and Receptor Mas Are Colocalized and Functionally Interdependent in Obese Zucker Rat Kidney

“…This notion is supported by our recent FRET studies in HEK cells expressing AT 2 R and MasR that their agonists alone do not affect FRET suggesting that these ligands do not promote or dissociate dimerization. 29 Along the same line of notion, other studies suggested that pretreatment of agonist or antagonist did not affect interactions of AT 2 R with AT 2 R, 36 AT 1 R 40 or B 2 R. 41 …”

“…36 Leonhardt and co-workers recently suggested that C35 of AT 2 R is essential for AT 2 R-MasR heterodimerization as FRET efficiency was found significantly decreased in HEK-293 cells transiently transfected with vectors encoding mutant AT 2 R-C35A-YFP and nonmutant MAS-CFP. 29 Particularly, Cys35 of the N terminus in one and Cys290 of exoloop 3 of other AT 2 R played a role in constitutive homodimerization of AT 2 R via disulfide bridge formation. 36 Another study demonstrated that deletion of residues 240–244 within intermediate portion of cytoloop 3 resulted in a complete loss of AT 2 R-mediated effects.…”

“…In support of such notion, molecular interactions between AT 2 R and MasR were demonstrated by fluorescence resonance energy transfer and cross relation spectroscopy in human kidney-293 cells transiently transfected with vectors encoding fluorophore-tagged AT 2 R and MasR. 29 …”

“…Functional inter-dependence observed in our study is in agreement with other studies showing abilities of AT 2 R antagonist to prevent the effects involving MasR and vice versa. 20–27,29,38,39 However, these in vivo and in vitro studies in isolated proximal tubule provide an interesting observation in that agonist activation of one receptor may not be critical for another receptor to function. This notion is supported by in vitro studies in isolated proximal tubules where either of the agonists alone AT 2 R agonist C21 or MasR agonist Ang-(1-7) was sufficient to increase NO formation.…”

Protective Angiotensin Type 2 Receptors in the Brain and Hypertension

“…Furthermore, there have been no studies which have assessed the cellular co-expression of Mas and AT2R in brain cardiovascular control centers. Nonetheless, a very recent article demonstrates dimerization between Mas and AT2R [54], and so their potential cooperation at the receptor level to lower blood pressure cannot be excluded.…”