Molecular cloning, sequencing and expression of an alpha 2-adrenergic receptor complementary DNA from rat brain

Abstract

We have isolated a cDNA clone from rat brain using a human platelet alpha 2-adrenergic receptor genomic clone as a probe. Comparison of the deduced amino acid sequence (450 residues) corresponding to the rat brain cDNA with that of the human platelet and human kidney alpha 2-adrenergic receptors showed 84% and 44% sequence similarity, respectively. The major sequence difference between the rat brain and human platelet proteins, was a stretch of 48 amino acids within the third cytosolic loop in which the similarity was only 42%. Analysis of the 48 amino acid-region indicated that the two receptors significantly differ in terms of their primary amino acid sequence and the predicted secondary and tertiary structural features. There was no sequence similarity between the human platelet and rat brain clone over the 177 bases of 3'-noncoding sequence and a less than 50% similarity over a stretch of 210 nucleotides in the 5'-untranslated region. Southern-blot analysis with a human platelet alpha 2-adrenergic receptor probe revealed the existence of a single 5.2 kb restriction fragment (KpnI/SacI) in both human and rat genomic DNA; the rat brain alpha 2-receptor probe, however, hybridized to a single 1.9 kb band in rat DNA. Northern-blot analysis of rat brain poly(A+) RNA with the rat brain cDNA probe under stringent hybridization conditions revealed a single 4.5 kb mRNA; none was detected by the human platelet receptor probe. The rat brain 4.5 kb mRNA was not detected in any (other than brain) tested rat tissues utilizing either rat brain or human platelet DNA probes. The rat brain cDNA was expressed in a mammalian cell line (COS-2A) and found to bind the alpha 2-adrenergic antagonist [3H]yohimbine; based on the binding-affinity for prazosin, the presently cloned receptor was pharmacologically closer to the alpha 2A subclass. We conclude that the rat brain cDNA encodes a new alpha 2-adrenergic receptor subtype that may be brain-specific.