Cloning, expression, and tissue distribution of the rat homolog of the bovine alpha 1C-adrenergic receptor provide evidence for its classification as the alpha 1A subtype.
Three alpha 1-adrenergic receptors (ARs) have been cloned, i.e., the alpha 1B-, alpha 1C-, and alpha 1D-ARs. Compared with the alpha 1B subtype, the alpha 1A subtype in tissue is described as being insensitive to chloroethylclonidine and sensitive to SZL-49 and having a 10-100-fold higher affinity for a number of agonists and antagonists. The alpha 1A subtype is also expressed in a variety of rat tissues (as assessed by pharmacology), with greatest abundance in the cerebral cortex, hippocampus, vas deferens, and submaxillary gland. The cloned bovine alpha 1C-AR, though having an alpha 1A-AR pharmacology, was first reported as not being expressed in any rat tissue (as determined by Northern analysis) and was therefore designated as a new subtype. We report the cloning, expression, and characterization of the rat homolog of the bovine alpha 1C-AR. Using a human alpha 1C-AR probe obtained by polymerase chain reaction screening of a neuroblastoma cell line (SK-N-MC), both exon 1 and exon 2 of the rat alpha 1C-AR gene were cloned from a rat genomic library. These two exons were spliced together and cloned into the expression vector pMT2'. Transfection into COS-1 cells and analysis of the ligand-binding profile of the expressed protein receptor using 125I-HEAT revealed a 10-100-fold higher affinity for the alpha 1-AR antagonists 5-methylurapidil, (+)-niguldipine, WB-4101, and phentolamine and the agonists oxymetazoline and methoxamine, compared with the alpha 1B-AR. This ligand-binding profile is similar to that for endogenously expressed tissue alpha 1A-ARs. In addition, the rat alpha 1C-AR was the least sensitive of the three cloned subtypes to the alkylating effects of chloroethylclonidine but was the most sensitive to the alkylating prazosin analog SZL-49, properties also observed for the tissue alpha 1A subtype. Furthermore, by three different techniques, i.e., RNase protection assays, reverse transcription-polymerase chain reaction Northern blotting, and in situ hybridization histochemistry, the rat alpha 1C-AR mRNA was localized to alpha 1A-AR-rich tissues, such as rat vas deferens, hippocampus, aorta, and submaxillary gland. Taken together, these data suggest that this receptor may actually represent the alpha 1A subtype.
Perez
D M
DM
Department of Molecular Cardiology, Cleveland Clinic Research Institute, Ohio 44195.
Piascik
M T
MT
Malik
N
N
Gaivin
R
R
Graham
R M
RM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D005091:Exons; D006801:Humans; D017403:In Situ Hybridization; D007438:Introns; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D018340:Receptors, Adrenergic, alpha-1 / Q000145:classification* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution
D012333:RNA, Messenger; D018340:Receptors, Adrenergic, alpha-1; D011994:Recombinant Proteins