Background-We previously identified the G-protein-coupled receptor Mas, encoded by the Mas proto-oncogene, as an endogenous receptor for the heptapeptide angiotensin-(1-7); however, the receptor is also suggested to be involved in actions of angiotensin II. We therefore tested whether this could be mediated indirectly through an interaction with the angiotensin II type 1 receptor, AT 1 . Methods and Results-In transfected mammalian cells, Mas was not activated by angiotensin II; however, AT 1 receptor-mediated, angiotensin II-induced production of inositol phosphates and mobilization of intracellular Ca 2ϩ was diminished by 50% after coexpression of Mas, despite a concomitant increase in angiotensin II binding capacity. Mas and the AT 1 receptor formed a constitutive hetero-oligomeric complex that was unaffected by the presence of agonists or antagonists of the 2 receptors. In vivo, Mas acts as an antagonist of the AT 1 receptor; mice lacking the Mas gene show enhanced angiotensin II-mediated vasoconstriction in mesenteric microvessels.Conclusions-These results demonstrate that Mas can hetero-oligomerize with the AT 1 receptor and by so doing inhibit the actions of angiotensin II. This is a novel demonstration that a G-protein-coupled receptor acts as a physiological antagonist of a previously characterized receptor. Consequently, the AT 1 -Mas complex could be of great importance as a target for pharmacological intervention in cardiovascular diseases.

Homo- and hetero-oligomerization of G-protein-coupled receptors (GPCRs) were examined in HEK-293 cells using two variants of bioluminescence resonance energy transfer (BRET). BRET(2) (a variant of BRET) offers greatly improved separation of the emission spectra of the donor and acceptor moieties compared with traditional BRET. Previously recorded homo-oligomerization of the human delta-opioid receptor was confirmed using BRET(2). Homo-oligomerization of the kappa-opioid receptor was observed using both BRET techniques. Both homo- and hetero-oligomers, containing both delta- and kappa-opioid receptors, were unaffected by the presence of receptor ligands. BRET detection of opioid receptor homo- and hetero-oligomers required expression of 50,000-100,000 copies of the receptor energy acceptor construct per cell. The effectiveness of delta-kappa-opioid receptor hetero-oligomer formation was as great as for homomeric interactions. The capacity of the two opioid receptors to form oligomeric complexes with the beta(2)-adrenoceptor was also assessed. Although such interactions were detected, at least 250,000 copies per cell of the energy acceptor were required. Requirement for high levels of receptor expression was equally pronounced in attempts to measure hetero-oligomer formation between the kappa-opioid receptor and the thyrotropin-releasing hormone receptor-1. These studies indicate that constitutively formed homo- and hetero-oligomers of opioid receptor subtypes can be detected in living cells containing less than 100,000 copies of the receptors. However, although hetero-oligomeric interactions between certain less closely related GPCRs can be detected, they appear to be of lower affinity than homo- or hetero-oligomers containing closely related sequences. Interactions recorded between certain GPCR family members in heterologous expression systems are likely to be artefacts of extreme levels of overexpression.

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The octapeptide hormone angiotensin (Ang) 2 II is one of the key components of the renin-angiotensin system and, as such, plays a major role in the regulation of blood pressure and cardiovascular homeostasis (1). Ang II exerts its effects through at least two subtypes of G proteincoupled receptors (GPCRs), the angiotensin II type 1 (AT 1 ) and 2 (AT 2 ) receptors. Whereas the functional role of the AT 2 receptor is not fully understood (2, 3), important biological functions such as vasoconstriction, salt/water reabsorption, and stimulation of aldosterone release are mediated through AT 1 receptor activation (4). However, in a number of situations, the AT 1 receptor is modulated by other coexpressed GPCRs. For example, interactions with the AT 2 receptor have been demonstrated in which the AT 2 receptor acts as a functional antagonist of the AT 1 receptor (5). AT 1 and bradykinin B 2 receptor interactions have also been shown, and these result in enhanced signaling of ligands at each receptor (6). Furthermore, in addition to interactions with GPCRs, agonist-induced functional interactions between the AT 1 receptor and the single transmembrane-spanning tyrosine kinase epidermal growth factor receptor have also been reported (7,8).The MAS proto-oncogene was first detected in vivo by its tumorigenic activity (9) and later identified as a member of the rhodopsin-like class A GPCR subfamily. However, although suggested in early studies to be a potential candidate as an Ang II receptor (10, 11), it remained an "orphan" until recently, when it was demonstrated that the Ang II metabolite Ang-(1-7) is an endogenous agonist of MAS (12). In the last decade, there has been emerging evidence that, although not able to bind or respond directly to Ang II, MAS has a physiological role in modulating the functions of Ang II in both the neuronal (13) and cardiovascular (14) systems. In MAS knock-out mice, AT 1 receptor signaling is altered in the amygdala (13) ; and, recently, Castro et al. (15) reported functional interactions between MAS and both the AT 1 and AT 2 receptors in mouse heart. In a previous study, we showed that MAS and the AT 1 receptor interact in heterologous expression systems and that MAS acts as an functional antagonist of the AT 1 receptor both in cotransfected CHO-K1 cells and in mesenteric microvessels of mice because greater levels of Ang II-mediated contraction were observed in vessels from MAS knock-out mice compared with wild-type controls (14). However, although Ang II produced lower levels of inositol phosphate accumulation and reduced increases in intracellular [Ca 2ϩ ] in CHO-K1 cells coexpressing MAS and the AT 1 receptor compared with those expressing the AT 1 receptor alone, we also observed that MAS coexpression with the AT 1 receptor resulted in an increase in [ 3 H]Ang II binding capacity (14). The mechanism by which MAS increases [ 3 H]Ang II binding has remained elusive. In this study, we demonstrate that it is due to the constitutive capacity of MAS to stimulate the G proteins G␣ q /G␣ 11...