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(1A), 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.