AFFINITY PURIFICATION OF ANGIOTENSIN TYPE-2 RECEPTORS FROM N1E-115 CELLS - EVIDENCE FOR AGONIST-INDUCED FORMATION OF MULTIMERIC COMPLEXES

The murine neuroblastoma N1E-115 cell line possesses type 1 and type 2 angiotensin II (AngII) receptor subtypes. In vitro differentiation of these cells substantially increases the density of the AT(2)-receptor subtype, whereas the density of the AT(1) receptors remains unchanged . In the present study, we report that the zwitterionic detergent chol amidopropyl)dimethylammonio]1-propanesulfonate (CHAPS) selectively sol ubilized AT(2) receptors from N1E-115 cell membranes and that these re ceptors could be purified further to near homogeneity by affinity chro matography. More specifically, the presence of an agonist (AngII) duri ng affinity purification of AT(2) receptors resulted in the elution of high (110-kDa) and low (66-kDa) molecular mass proteins as determined by gel electrophoresis under nonreducing conditions. In contrast, whe n the nonselective antagonist Sar(1),IIe(8)-AngII was used during puri fication, only the lower 66-kDa protein was observed. Affinity purific ation in the presence of the peptide and nonpeptide AT(2)-receptor ant agonists CGP42112A and PD123319 also resulted in elution of the same 6 6-kDa protein, but unlike that in the presence of Sar(1),IIe(8)-AngII, some of the high molecular weight site was observed as well. On the o ther hand, Losartan, an AT(1)-receptor antagonist, was completely inef fective in eluting any AngII receptors from the affinity column, furth er confirming their AT(2) identity. After agonist elution, the 110-kDa band dissociated into two low molecular mass bands of 66 kDa and 54 k Da when sodium dodecyl sulfate-gel electrophoresis was run under reduc ing conditions. The 110-kDa and 66-kDa proteins, but not smaller, affi nity purified proteins, specifically bound I-125-AngIl as determined b y covalent cross-linking of I-125-AngII to the receptors with the homo bifunctional cross-linker disuccinimidyl suberate, or by size exclusio n chromatography on a TSK 3000 SW column. Lastly, immunoblot analysis of affinity-purified material with antibodies selective for AT(2) rece ptors revealed major immunoreactive proteins of 110 kDa and 66 kDa in the presence of an agonist, whereas the same 66-kDa protein, as well a s a smaller (54-kDa) immunoreactive protein, was detected under reduci ng conditions. Collectively, these data suggest that CHAPS-solubilized AT(2) receptors from N1E-115 cells may consist of a binding protein o f approximately 66 kDa, which in the presence of an agonist readily as sociates with other smaller proteins to form larger multimeric complex es.