Imidazoline Receptor Antisera-Selected (IRAS) cDNA: Cloning and Characterization

Piletz, John E.; Ivanov, Tina R.; Sharp, John D.; Chang, Chung-Ho; Pickard, Richard T.; Ernstmerger, Paul; Gold, Gerry
June 2000
DNA & Cell Biology;Jun2000, Vol. 19 Issue 6, p319
Academic Journal
The imidazoline-1 receptor (IR[sub 1]) is considered a novel target for drug discovery. Toward cloning an IR[sub 1], a truncated cDNA clone was isolated from a human hippocampal lambda gt11 cDNA expression library by relying on the selectivity of two antisera directed against candidate IR proteins. Amplification reactions were performed to extend the 5 ' and 3 ' ends of this cDNA, followed by end-to-end PCR and conventional cloning. The resultant 5131-basepair molecule, designated imidazoline receptor-antisera-selected (IRAS) cDNA, was shown to encode a 1504-amino acid protein (IRAS-1). No relation exists between the amino acid sequence of IRAS-1 and proteins known to bind imidazolines (e.g., it is not an alpha [sub 2]-adrenoceptor or monoamine oxidase subtype). However, certain sequences within IRAS-1 are consistent with signaling motifs found in cytokine receptors, as previously suggested for an IR[sub 1]. An acidic region in IRAS-1 having an amino acid sequence nearly identical to that of ryanodine receptors led to the demonstration that ruthenium red, a dye 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[sub 1] sites in untransfected PC-12 cells (a source of authentic I[sub 1] binding sites). 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-specific manner, transfection of IRAS cDNA into Chinese hamster ovary cells led to high-affinity I[sub 1] binding sites by criteria of nanomolar affinity for moxonidine and rilmenidine. Thus, IRAS1 is the first protein discovered with characteristics of an IR[sub 1].


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