SOLUTION-PHASE LIBRARY SCREENING FOR THE IDENTIFICATION OF RARE CLONES - ISOLATION OF AN ALPHA-1D-ADRENERGIC RECEPTOR CDNA

Alpha-1-Adrenergic receptor (alpha-1-AR) Subtypes (alpha-1A and alpha- 1B) play a critical role in vascular smooth muscle contraction and cir culatory homeostasis. Transcripts for these guanine nucleotide-binding protein-coupled receptors are extremely low in abundance, however, an d isolation of their cDNAs is difficult. We have developed a novel tec hnique for identifying rare clones in a cDNA library, which has been u sed successfully to isolate a cDNA clone encoding an alpha-1D-AR. A 56 4-bp polymerase chain reaction product encoding a region between the t hird and sixth transmembrane domains of the alpha-1D-AR was first gene rated using rat brain mRNA as template and highly degenerate primers. The primers corresponded to those domains but contained mismatches to the alpha-1B-AR sequences. A 3-kb transcript was identified with this polymerase chain reaction probe, by Northern analysis of rat hippocamp us. However, traditional plaque hybridization failed to identify a cDN A in a rat hippocampus lambda-gt10 library. By solution-phase screenin g of virtually the entire library, a cDNA containing a 3-kb insert was identified, amplified, and purified. This insert encodes a 560-amino acid protein corresponding to the topology of guanine nucleotide-bindi ng protein-coupled receptors. This receptor has approximately 71% amin o acid identity, in the transmembrane regions, to the hamster and rat alpha-1B-ARs. Characterization of the receptor expressed in COS-7 cell s, by ligand binding and photoaffinity labeling, revealed some of the characteristics of an alpha-1A-AR. However, unlike alpha-1A-ARs charac terized previously in membrane preparations or in solubilized partiall y purified preparations, the expressed receptor Could be extensively i nactivated by chlorethylclonidine. in addition, it displays ligand-bin ding properties that are not consistent with an alpha-1A-AR. This indi cates that the cDNA clone that we have isolated encodes a novel alpha- 1-AR subtype, which we classify as the alpha-1D-AR.