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 AT2-receptor subtype, whereas the density of the AT1 receptors remains unchanged. In the present study, we report that the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) selectively solubilized AT2 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 (AngII) during affinity purification of AT2 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 Sar1,Ile8-AngII was used during purification, only the lower 66-kDa protein was observed. Affinity purification in the presence of the peptide and nonpeptide AT2-receptor antagonists CGP42112A and PD123319 also resulted in elution of the same 66-kDa protein, but unlike that in the presence of Sar1,Ile8-AngII, some of the high molecular weight site was observed as well. On the other hand, Losartan, an AT1-receptor antagonist, was completely ineffective in eluting any AngII receptors from the affinity column, further confirming their AT2 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Siemens
I R
IR
Department of Animal Biology, University of Pennsylvania, Philadelphia 19104.
Yee
D K
DK
Reagan
L P
LP
Fluharty
S J
SJ
D000804:Angiotensin II / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002793:Cholic Acids; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008970:Molecular Weight; D009447:Neuroblastoma; D011945:Receptors, Angiotensin / Q000302:isolation & purification* / Q000378:metabolism*; D013388:Succinimides; D014407:Tumor Cells, Cultured
D002793:Cholic Acids; D003432:Cross-Linking Reagents; D003902:Detergents; D046911:Macromolecular Substances; D011945:Receptors, Angiotensin; D013388:Succinimides; D000804:Angiotensin II; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; C019358:disuccinimidyl suberate