Imidazoline receptor antisera-selected (IRAS) cDNA: cloning and characterization.
The imidazoline-1 receptor (IR1) is considered a novel target for drug discovery. Toward cloning an IR1, 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 alpha2-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 IR1. 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 I1 sites in untransfected PC-12 cells (a source of authentic I1 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 I1 binding sites by criteria of nanomolar affinity for moxonidine and rilmenidine. Thus, IRAS-1 is the first protein discovered with characteristics of an IR1.
Piletz
J E
JE
Department of Psychiatry, University of Mississippi Medical Center, Jackson 39216-4505, USA. jpiletz@psychiatry.umsmed.edu
Ivanov
T R
TR
Sharp
J D
JD
Ernsberger
P
P
Chang
C H
CH
Pickard
R T
RT
Gold
G
G
Roth
B
B
Zhu
H
H
Jones
J C
JC
Baldwin
J
J
Reis
D J
DJ
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells / Q000378:metabolism; D019556:COS Cells / Q000378:metabolism; D003000:Clonidine / Q000031:analogs & derivatives / Q000378:metabolism; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary; D004837:Epinephrine / Q000378:metabolism; D006801:Humans; D019329:Idazoxan / Q000378:metabolism; D007093:Imidazoles / Q000378:metabolism; D054755:Imidazoline Receptors; D007106:Immune Sera; D007457:Iodine Radioisotopes; D008969:Molecular Sequence Data; D009278:Naphazoline / Q000378:metabolism; D011955:Receptors, Drug / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism*; D012430:Ruthenium Red / Q000737:chemistry / Q000378:metabolism; D016324:Sequence Tagged Sites; D013194:Staining and Labeling; D014162:Transfection; D015016:Yohimbine / Q000378:metabolism
D018076:DNA, Complementary; D007093:Imidazoles; D054755:Imidazoline Receptors; D007106:Immune Sera; D007457:Iodine Radioisotopes; D011955:Receptors, Drug; C410887:imidazoline I1 receptors; C053113:4-iodoclonidine; D012430:Ruthenium Red; D015016:Yohimbine; C043482:moxonidine; D009278:Naphazoline; D003000:Clonidine; D019329:Idazoxan; D004837:Epinephrine