CONTRIBUTION OF A 12-KDA PROTEIN TO THE ANGIOTENSIN-II-INDUCED STABILIZATION OF ANGIOTENSINOGEN MESSENGER-RNA - INTERACTION WITH THE 3' UNTRANSLATED MESSENGER-RNA

Several authors have shown that angiotensin II stimulates hepatic angi otensinogen synthesis in vivo, ex vivo and in vitro. In previous studi es we have demonstrated that this effect of angiotensin II depends mai nly on a transient inhibition of adenylyl cyclase and is the consequen ce of a stabilization of angiotensinogen mRNA. In the present study we describe the isolation of a polysomal 12 kDa protein which, in band s hift and cross link assays, shows a specific affinity to the 3' untran slated region (3' UTR) of angiotensinogen mRNA and prevents enzymatic degradation of angiotensinogen mRNA in a cell-free incubation system. [P-32]UTP-labelled or unlabelled 3' fragments of angiotensinogen mRNA were synthesized on a transcription vector (pGEM5zf+) into which the c orresponding DNA sequence was cloned after restriction from vector pRA G 16. Binding of the 12 kDa protein to the radioactively labelled 3' U TR of angiotensinogen mRNA could be displaced by unlabelled 3' UTR mRN A fragments but not by a renin mRNA of comparable length derived from the coding region. The RNA-binding protein appears to be derived from a higher molecular mass precursor (45 kDa) which is preferentially pre sent under reducing conditions in vitro; the active low molecular mass form is evident in the absence of reducing agents. In a cross link ex periment we established that a band shift signal which was obtained in the presence of the 45 kDa protein preparation exclusively depends on RNA binding of the active 12 kDa protein. In addition, a phosphorylat ion step may be involved in the activation of the 12 kDa protein, sinc e its molecular mass and isoelectric point correlate with proteins whi ch were phosphorylated in response to transient decreases of cAMP (ind uced by guanfacine or angiotensin II) or in response to a direct inhib ition of protein kinase A by the cAMP antagonist Rp-cAMP. The importan ce of phosphorylation reactions for the stabilization of angiotensinog en mRNA was further assessed in a cell-free incubation system of rat l iver parenchymal cells. These studies demonstrated that in the presenc e of acid phosphatase (1 U/ml) the half-life of angiotensinogen was si gnificantly decreased. In the same incubation system the 12 kDa protei n increased the half-life of endogenous as well as of exogenous angiot ensinogen mRNA three- to fourfold, while no stabilizing effect was app arent when exogenous angiotensinogen mRNA from which the 3' tail had b een deleted was added. We concluded that an intracellular 12 kDa prote in may play a crucial role in the angiotensin II-induced stabilization of hepatic angiotensinogen mRNA and further suggest that this protein exerts its effect via binding to the 3' UTR of angiotensinogen mRNA i n response to a cAMP-dependent activation step.