Angiotensin II AT2 receptors are functionally coupled to protein tyrosine dephosphorylation in N1E-115 neuroblastoma cells.
Murine N1E-115 neuroblastoma cells are shown to express a single class of angiotensin II (Ang II) receptors that display all the pharmacological properties defining the Ang II receptor subtype 2 (AT2): high affinity for 125I-labelled AT2-selective agonist CGP 42112 (Kd 91 +/- 19 pM); expected rank order of potency (CGP 42112 = (Sar1,Ile8)Ang II > or = Ang II > PD 123319 >> DUP 753) for several Ang II analogues; increased binding in the presence of the reducing reagent dithiothreitol (DTT); and insensitivity to analogues of GTP. Molecular cloning of cDNA encoding AT2 receptors from N1E-115 cells reveals nucleotide sequence identity with the AT2 subtype expressed in fetal tissue. Murine AT2 receptors transiently expressed in COS cells display the same pharmacological profile as endogenous Ang II receptors of N1E-115 cells. Taken together, these data reveal the exclusive presence of the AT2 receptor subtype in N1E-115 cells. Incubation of N1E-115 cells with Ang II leads to a marked decrease in the level of tyrosine phosphorylation of several proteins with apparent molecular masses of 80, 97, 120, 150 and 180 kDa respectively. Tyrosine dephosphorylation of the same set of proteins is observed after treatment with the AT2-specific agonist CGP 42112. The response to both effectors is rapid and transient, showing a maximum between 5 and 10 min, and returning to basal levels after 20-30 min. In both cases, tyrosine dephosphorylation can be prevented by co-incubation with an excess of the antagonist Sarile. These data thus establish that AT2 receptor activation leads to protein tyrosine dephosphorylation in N1E-115 cells, and support a possible role for AT2 receptors in the negative regulation of cell proliferation.
Nahmias
C
C
Institut Cochin de Génétique Moléculaire, CNRS UPR 0415, Paris, France.
Cazaubon
S M
SM
Briend-Sutren
M M
MM
Lazard
D
D
Villageois
P
P
Strosberg
A D
AD
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004229:Dithiothreitol / Q000494:pharmacology; D015870:Gene Expression; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D009447:Neuroblastoma / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011945:Receptors, Angiotensin / Q000235:genetics / Q000502:physiology*; D014162:Transfection; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D018076:DNA, Complementary; D009842:Oligopeptides; D011945:Receptors, Angiotensin; C060894:CGP 42112A; D019000:Phosphotyrosine; D014443:Tyrosine; D006160:Guanosine Triphosphate; D011505:Protein-Tyrosine Kinases; D004229:Dithiothreitol