Premium
Affinity Purification of Angiotensin Type 2 Receptors from N1E‐115 cells: Evidence for Agonist‐Induced Formation of Multimeric Complexes
Author(s) -
Siemens Ivo R.,
Yee Daniel K.,
Reagan Lawrence P.,
Fluharty Stever J.
Publication year - 1994
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1994.62010257.x
Subject(s) - receptor , losartan , agonist , chemistry , angiotensin ii , gel electrophoresis , angiotensin ii receptor type 1 , molecular mass , affinity chromatography , chromatography , peptide , biochemistry , enzyme
The murine neuroblastoma N1E‐115 cell line possesses type 1 and type 2 angiotensin II (Angll) 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 3‐[(3‐cholamidopropyl)dimethylammonio]‐ 1‐propanesulfonate (CHAPS) selectively solubilized AT 2 receptors from N1E‐115 cell membranes and that these receptors could be purified further to near homogeneity by affinity chromatography. More specifically, the presence of an agonist (Angll) during 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, when the nonselective antagonist Sar 1 , lle 8 ‐Angll was used during purification, only the lower 66‐kDa protein was observed. Affinity purification in the presence of the peptide and nonpeptide AT 2 ‐receptor antagonists CGP42112A and PD123319 also resulted in elution of the same 66‐kDa protein, but unlike that in the presence of Sar 1 , lle 8 ‐Angll, some of the high molecular weight site was observed as well. On the other hand, Losartan, an AT 1 ‐receptor antagonist, was completely ineffective in eluting any Angll receptors from the affinity column, further confirming their AT 2 identity. After agonist elution, the 110‐kDa band dissociated into two low molecular mass bands of 66 kDa and 54 kDa when sodium dodecyl sulfate‐gel electrophoresis was run under reducing conditions. The 110‐kDa and 66‐kDa proteins, but not smaller, affinity‐purified proteins, specifically bound 125 l‐Angll as determined by covalent cross‐linking of 125 l‐Angll to the receptors with the homobifunctional cross‐linker disuccinimidyl suberate, or by size exclusion chromatography on a TSK 3000 SW column. Lastly, immunoblot analysis of affinity‐ purified material with antibodies selective for AT 2 receptors revealed major immunoreactive proteins of 110 kDa and 66 kDa in the presence of an agonist, whereas the same 66‐kDa protein, as well as a smaller (54‐kDa) immunoreactive protein, was detected under reducing conditions. Collectively, these data suggest that CHAPS‐solubilized AT 2 receptors from N1E‐115 cells may consist of a binding protein of approximately 66 kDa, which in the presence of an agonist readily associates with other smaller proteins to form larger multimeric complexes.