Imidazoline receptor antisera-selected (IRAS) cDNA: Cloning and characterization

The imidazoline-1 receptor (IR1) is considered a novel target for drug disc overy. Toward cloning an IR1, a truncated cDNA clone was isolated from a hu man hippocampal lambda gt11 cDNA expression library by relying on the selec tivity of two antisera directed against candidate IR proteins. Amplificatio n reactions were performed to extend the 5' and 3' ends of this cDNA, follo wed by end-to-end PCR and conventional cloning. The resultant 5131-basepair molecule, designated imidazoline receptor-antisera-selected (IRAS) cDNA, w as shown to encode a 1504-amino acid protein (IRAS-1). No relation exists b etween the amino acid sequence of IRAS-1 and proteins known to bind imidazo lines (e.g., it is not an alpha(2)-adrenoceptor or monoamine oxidase subtyp e). However, certain sequences within IRAS-1 are consistent with signaling motifs found in cytokine receptors, as previously suggested for an IR1. An acidic region in IRAS-1 having an amino acid sequence nearly identical to t hat of ryanodine receptors led to the demonstration that ruthenium red, a d ye that binds the acidic region in ryanodine receptors, also stained IRAS-1 as a 167-kD band on SDS gels and inhibited radioligand binding of native I -1 sites in untransfected PC-12 cells (a source of authentic I1 binding sit es). Two epitope-selective antisera were also generated against IRAS-1, and both reacted with the same 167-kD band on Western blots. In a host-cell-sp ecific manner, transfection of IRAS cDNA into Chinese hamster ovary cells l ed to high-affinity I1 binding sites by criteria of nanomolar affinity for moxonidine and rilmenidine. Thus, IRAS-1 is the first protein discovered wi th characteristics of an IR1.