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A good in vitro model within which to investigate molecular interactions between feeding relevant neuropeptide systems has been lacking. Consequently, we began using reverse transcriptase-polymerase chain reaction (RT-PCR) to screen various neuronal cell lines for the presence of feeding relevant neuropeptides and receptors. N1E-115 murine neuroblastoma cells have emerged as an attractive candidate for further analysis because they contain mRNA for a variety of key systems implicated in the regulation of energy homeostasis., (Copyright 2002 Elsevier Science B.V.)

Key amino terminal residues in type 1 (AT1) angiotensin II (AngII) receptors are not conserved within type 2 (AT2) receptors. We therefore characterized amino terminal mutants that are transiently expressed in COS-3 membranes. AT2 amino terminal deletion drastically reduced affinity for AngII, suggesting its importance for this subtype. AT1-AT2 amino terminal exchanges retained wild type AngII affinities (Kd ranging from 3-5 nM), indicating compensation despite substantial sequence dissimilarities. Finally, binding of AT2 selective ligands (CGP42112A and PD123319) was not dependent on the amino terminus.

A fifth transmembrane domain lysine residue is conserved in both the type 1 (AT1) and type 2 (AT2) angiotensin II (AngII) receptors. This lysine (Lys199) is believed to play a critical role in peptide binding for the AT1 receptor. To evaluate its possible role in the AT2 receptor, the analogous AT2 residue (Lys199) was changed to glutamine. This mutation greatly reduced the affinity for both 125I-AngII and 125I-Sar1,Ile8-AngII and abolished binding to the non-peptide 125I-PD122979. These data indicate that despite a relatively low homology of 34%, some commonalities in the binding mechanism for AngII may exist between the two subtypes.

Homology-based PCR was used to isolate angiotensin II type 2 (AT2) receptor cDNA from murine neuroblastoma N1E-115 cells. Despite subtle differences in the nucleotide sequence (the N1E-115 clone coded for Phe133 as TTC and Gln326 as CAG; base substitutions are in bold-italics), the AT2 receptor protein was identical to other reported murine AT2 clones. When transfected into COS-1 cells, the expressed AT2 receptor displayed high affinity for AngII and for AT2-selective compounds, GTP gamma S-insensitive agonist binding and enhanced agonist binding by dithiothreitol. Previously, we have demonstrated that N1E-115 cells possess two distinct subpopulations of AT2 receptors, defined as peak I and peak III receptors, that can be separated by heparin-sepharose chromatography. The two subpopulations differ pharmacologically, biochemically and immunologically. The binding properties of the cloned AT2 receptor closely resembled that of peak III receptors. Moreover, antisera raised against peak I AT2 receptors failed to immunoreact to either peak III receptors or cloned AT2 receptors expressed in COS-1 cells. Collectively, these data suggest that the cloned AT2 receptor is identical to peak III receptors from N1E-115 cells and that a novel AT2 receptor (peak I) remains to be cloned.