Cloning, expression, and tissue distribution of the rat homolog of the bovine α_1C-adrenergic receptor provide evidence for its classification as the α_1A subtype

Three α₁-adrenergic receptors (ARs) have been cloned, i.e., the α_1B-, α_1C-, and α_1D-ARs. Compared with the α_1B subtype, the α_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 α_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 α_1C-AR, though having an α_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 α_1C-AR. Using a human α_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 α_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 α₁-AR antagonists 5-methylurapidil, (+)-niguldipine, WB-4101, and phentolamine and the agonists oxymetazoline and methoxamine, compared with the α_1B-AR. This ligand-binding profile is similar to that for endogenously expressed tissue α_1A-ARs. In addition, the rat α_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 α_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 α_1C-AR mRNA was localized to α_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 α_1A subtype.