The renin angiotensin system is important in cardiovascular regulation and angiotensin II (ang II) is its primary mediator. Ang II activates receptors that are important in control of responsiveness but which have been studied largely indirectly, in contrast with many other receptors for which primary amino acid structure is known. The ang II receptor has been difficult to purify and clone. We now have isolated from a rat aortic cDNA library a clone that encodes a receptor with characteristics of the vascular smooth muscle ang II receptor. In COS cells transfected with this clone, [125I] Sar1-ile8-ang II binding is saturable with a Kd of 0.3 nM, and the receptor has an appropriate potency series for binding inhibition of ang II>ang III>ang I, with an IC50 for ang II of 1.6 nM. The nonpeptidic """"""""Type 1"""""""" ang II receptor antagonist DuP753 competes potently (IC50 6.2 nM), whereas the """"""""Type 2"""""""" antagonist PD123177 does not compete. These features of the transfected receptor are identical to those of the vascular receptor. We shall now exploit this advance to extend our previous work on the secondary signalling mechanisms of the ang II receptor in vascular smooth muscle to explore directly the structure-function relationships. Several areas will be investigated. First, we will examine how the ang II receptor relates to the superfamily of G-protein coupled receptors, since it is also coupled to phospholipase C through a G-protein. Second, we will attempt to account for the considerable heterogeneity of ang II-mediated pharmacological responses putatively attributed to the existence of more than one of the major observations of the current grant period - namely, that the vascular ang II receptor activates sequentially a phospholipase C and a phospholipase D, implying different phasic and tonic signalling mechanisms. This issue relates to the general question of how one receptor couples to different effector mechanisms. To achieve these goals we propose three Specific Aims: 1. To characterize rigorously the cDNA clone that we have isolated and to classify it structurally within the superfamily of G-protein coupled receptors; 2. To identify and characterize other ang II receptor subtypes; and 3. To define the structural determinants of ang II receptor coupling to different effector phospholipases. These data will provide important insights into molecular mechanisms of action of ang II and should have important implications for understanding the normal control of blood pressure and hypertension. In particular, it should now be possible to address the issue of whether the ang II receptor is a candidate gene contributing to hypertension.
Showing the most recent 10 out of 14 publications
