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 insens itive 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 a ssessed by pharmacology), with greatest abundance in the cerebral cort ex, hippocampus, vas deferens, and submaxillary gland. The cloned bovi ne alpha(1C)-AR, though having an alpha(1A)-AR pharmacology, was first reported as not being expressed in any rat tissue (as determined by N orthern analysis) and was therefore designated as a new subtype. We re port 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 (S K-N-MC), both exon 1 and exon 2 of the rat alpha(1C)-AR gene were clon ed from a rat genomic library. These two exons were spliced together a nd cloned into the expression vector pMT2'. Transfection into COS-1 ce lls and analysis of the ligand-binding profile of the expressed protei n receptor using I-125-HEAT revealed a 10-100-fold higher affinity for the alpha(1)-AR antagonists 5-methylurapidil, (+)-niguldipine, WB-410 1, and phentolamine and the agonists oxymetazoline and methoxamine, co mpared with the alpha(1B)-AR. This ligand-binding profile is similar t o that for endogenously expressed tissue alpha(1A)-ARs. In addition, t he rat alpha(1C)-AR was the least sensitive of the three cloned subtyp es to the alkylating effects of chloroethylclonidine but was the most sensitive to the alkylating prazosin analog SZL-49, properties also ob served for the tissue alpha(1A) subtype. Furthermore, by three differe nt techniques, i.e., RNase protection assays, reverse transcription-po lymerase chain reaction Northern blotting, and in situ hybridization h istochemistry, the rat alpha(1C)-AR mRNA was localized to alpha(1A)-AR -rich tissues, such as rat vas deferens, hippocampus, aorta, and subma xillary gland. Taken together, these data suggest that this receptor m ay actually represent the alpha(1A) subtype.