The central actions of angiotensin II (AngII) are importantly involved in the regulation of cardiovascular homeostasis. However, relatively little is known about the biochemical and molecular properties of neuronal AngII receptors (AngII-Rs), in part because they are located in very small, restricted regions of the brain. Much of what is known about AngII-Rs is based on studies using peripheral target tissues. However, recent evidence suggests that there are multiple subtypes of AngII-Rs present in the brain, and that at least one of the these receptor subtypes is unique to the nervous system. Therefore, inferences drawn from studies of peripheral receptors are unlikely to be applicable to AnglI-R expression in the brain. Previously, we have reported that the murine neuroblastoma N1E-115 cell line is a very useful model system for the molecular analysis of AngII-Rs insofar as it expresses functional AngII-R subtypes that appear identical to those found in the brain. At present it is not possible to determine if the apparent multiplicity of AngII-Rs in the nervous system results from the activity of unique genes, or, alternatively, cell-specific post-translational processing of a single gene product. The only definitive way to address this issue is to clone the cDNAs encoding for AngII-Rs. In view of the high density and multiplicity of AngII-R subtypes expressed in N1E-115 cells, these cells should prove to be an ideal cell culture system in which to pursue such investigations. Accordingly, the major goal of the present application is to isolate the cDNAs encoding for the multiple subtypes of AngII-Rs expressed in N1E-115 cells by using three complimentary approaches. These approaches will include the homology-based technique of PCR using primers derived from the superfamily of G-protein-coupled receptors, antibody screening of our cDNA library and expression cloning in oocytes. Once these cDNA clones are obtained we will determine their functional properties after transfecting them in AtT-20 cells. In particular, we will investigate the hypothesis that each of the cDNAs encodes for a unique subtype of AngII-R, which, in turn, is differentially coupled to specific intracellular effectors such as phopshoinositide hydrolysis and guanylate cyclase, and that these transduction pathways interact during agonist-induced desensitization. Finally, we will examine the regulation of mRNA levels for each AngII-R subtype during cellular differentiation and following treatment with the mineralocorticoid, aldosterone. Collectively, these experiments should provide important information about the transcriptional and posttranslational relationships amongst the subtypes of neuronal AngII-Rs, the functional properties of each subtype, and the capacity for selective regulation of AngII-R subtype expression in the nervous system.
