CLONING AND EXPRESSION OF A RAT-BRAIN ALPHA-2B-ADRENERGIC RECEPTOR

We have isolated a cDNA clone (RB-alpha-2B) and its homologous gene (GR-alpha-2B) encoding an alpha-2B-adrenergic receptor subtype by screening a rat brain cDNA and a rat genomic library. Nucleotide sequence analysis showed that both clones code for a protein of 458 amino acids, which is 87% homologous to the human kidney glycosylated adrenergic receptor (alpha-2-C4) and divergent from the rat kidney nonglycosylated alpha-2B subtype (RNG-alpha-2). Transient expression of RB-alpha-2B in COS-7 cells resulted in high-affinity saturable binding (Kd = 0.25 nM) for [H-3]rauwolscine and a high receptor number (Bmax = 7.7 pmol/mg of protein) in the membranes of transfected COS-7 cells. Pharmacological analysis demonstrated that the expressed receptor bound adrenergic ligands with the following order of potency: rauwolscine > yohimbine > prazosin > oxymetazoline, with a prazosin-to-oxymetazoline Ki ratio of 0.34. This profile is characteristic of the alpha-2B-adrenergic receptor subtype. Blotting analysis of rat brain mRNA gave one major (3.0-kilobase) and two minor (4.6- and 2.3-kilobase) mRNA species, and hybridization with strand-specific probes showed that both DNA strands of GR-alpha-2B may be transcriptionally active. These findings show that rat brain expresses an alpha-2B-adrenergic receptor subtype that is structurally different from the rat kidney nonglycosylated alpha-2B subtype. Thus the rat expresses at least two divergent alpha-2B-adrenergic receptors.