DEVELOPMENT OF POLYCLONAL ANTIBODIES AGAINST ANGIOTENSIN TYPE-2 RECEPTORS

Murine neuroblastoma N1E-115 cells are a useful system in which to stu dy neuronal angiotensin II (AngII) receptors. N1E-115 cells possess bo th type 1 (AT1) and type 2 (AT2) AngII receptor subtypes, as does mamm alian brain. AT2 receptors in brain or N1E-115 cells can be solubilize d in 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate. In th e present study, heparin-Sepharose chromatography was used to partiall y purify solubilized N1E-115 membranes to produce an enriched populati on of AT2 receptors. Subsequently, an eluted peak, containing the majo rity of AT2 binding activity, was used as an immunogen in the developm ent of protein polyclonal antibodies. The antibodies specifically dete cted immunoreactive proteins of approximately 110 and 66 kDa in both s olubilized N1E-115 cells, as well as the original protein material tha t eluted from the heparin-Sepharose column, whereas no such immunoreac tivity was detected in a kidney epithelial cell line that lacks any sp ecific I-125-labeled AngII (I-125-AngII) binding activity. Moreover, t he antibodies immunoreacted with affinity-purified AT2 receptors. Thes e antibodies were also able to immunoprecipitate AT2 receptors from so lubilized N1E-115 cells, as revealed by the pharmacologic profile of I -125-AngII binding to the precipitated protein. Similarly, the antibod ies were able to immunoprecipitate a 66-kDa protein that had been cova lently crosslinked with I-125-AngII by use of the homobifunctional cro sslinker dithiobis(succinimidyl propionate). Collectively, these resul ts demonstrate the development of a specific AT2 receptor antibody tha t may be used to further characterize this receptor subtype at both th e cellular and molecular levels.