Recent discoveries of a (pro)renin receptor ((P)RR) and the monocarboxypeptidase angiotensin-converting enzyme-2 (ACE-2) have revolutionized thinking about the renin-angiotensin system (RAS) and cardiovascular disease (CVD). Not only does the (P)RR signal in response to renin and prorenin, it also activates prorenin and increases the ability of renin to form angiotensin I. ACE-2 metabolizes angiotensin II (AngII) to form Ang-(1-7), an agonist of the deorphanized G protein-coupled receptor mas, (ACE-2/Ang-(1-7)/mas axis). Ang-(1-7) exerts depressor, antifibrotic and anti-inflammatory actions that counteract the pathophysiological effects of Ang II on the cardiovascular system mediated by the AT1 angiotensin receptor. ACE-2's dual function: to inactivate AngII and form a physiological antagonist of AngII, makes it an exciting therapeutic target for treating CVD associated with overactivity of the AngII/AT1 receptor axis. This proposal will develop radioligands to measure functional (P)RR binding and ACE-2 activity that complement current assay methodologies. The (P)RR radioligand will be prorenin, radioiodinated using iodogen with resolution of the radioiodinated protein from unreacted iodine-125 by ion-exchange chromatography. The ACE-2 radioligand is an analog of MLN-4760, a drug initially developed to inhibit ACE-2. The hypotheses of this proposal are: 1) that radioiodinated prorenin can identify and measure (P)RR in rodent tissues, and 2) that an MLN-4760 analog in which a leucinyl side chain is replaced with a tyrosinyl side chain near the carboxy-terminal of this pseudodipeptide will retain the high affinity and specificity of MLN-4760 for ACE-2, with and without iodine-125 on the tyrosinyl side chain. Proposed studies involve 1) Radioiodination of prorenin, and 2) MLN-4760 analog and HPLC purification of the mono125I (as well as non-radioactive mono127I) analog of MLN-4760, 3) use of these radioligands to measure (P)RR and ACE-2 in brain and kidney by radioligand binding and autoradiography, 4) assessment of inhibition of ACE-2 activity by the analog using a fluorogenic assay.