An a2-adrenergic receptor subtype has been isolated from a human genomic spleen library using the human 5-hydroxytryptaminelA receptor gene (also known as G-21) as a probe. This adrenergic receptor gene encodes a protein of 450 amino acids and does not contain any consensus sequences for N-linked glycosylation in its amino terminus or extracellular loops. This receptor is also distinguished by the presence of 12 con-secutive glutamic acid residues in the region of its third intracellular loop. The deduced amino acid sequence shows greatest homology to previously cloned human cx2-adrenergic receptors and has structural similarities to other guanine nucleotide-binding protein-coupled receptors. The DNA encoding the human a2 receptor was stably transfected into mouse fibroblast Ltk cells and radioligand binding studies were performed using the a2 antagonist[3H] rauwolscine.[3H] Rauwolscine bound with high affinity (Kd 0.33 nM) and in a saturable manner(Bmax 1.4 pmol/mg of protein). Pharmacological characterization of this receptor indicated a rank order of potency of yohimbine> prazosin> oxymetazoline. Additionally, 100 MM 5’-guanylylimi-dodiphosphate, produced a rightward shift in the epinephnne competition curve, with resultant increases in both the K, value and Hill coefficient, suggestive of a functional interaction of the cloned receptor with native guanine nucleotide-binding protein (s) of Ltk membranes. The data presented here are consistent with previous biochemical and pharmacological studies on a2 receptors and are supportive of the designation of this receptor as an a2B subtype.

Radioligand binding studies strongly point toward the existence of at least three cs2-adrenergic receptor subtypes, designated a2A, a2B, and asc (1). These receptors belong to the family of G protein-coupled receptors that are distinguished by their seven transmembrane-spanning-region configuration and their functional coupling to effector mechanisms via distinct G pro-teins (2). All three subtypes display a high affinity for the a2 antagonists yohimbine and rauwolscine but differ in their affinities for various drugs, which can differentiate between a2-adrenergic subtypes(1). Oxymetazoline exhibits high affinity for the a2A subtype, whereas prazosin displays high affinity for the asB subtype(3). The a2c subtype is pharmacologically similar to a2B, but a (has a higher affinity (‘-10-fold) for [3H] rauwolscine than the a28 subtype(4). DNA sequences coding for two human a2-adrenergic recep-tors have been isolated. A receptor from human platelet has been localized to chromosome 10, having clear pharmacological characteristics of an a2A receptor(5), and has been designated a2-C10 (6). Another receptor, cloned from human kidney, has been localized to chromosome 4 and is called a2-C4 (6). The presence of a third a2 subtype residing on human chromosome 2 has been implied from Southern blot analysis(5) and has not been previously cloned.