Cloning and expression of the alpha 2C-adrenergic receptor from the OK cell line.

The alpha 2-adrenergic receptors have been divided into four pharmacological subtypes, alpha 2A, alpha 2B, alpha 2C, and alpha 2D. The OK cell line, a cell line derived from an opossum kidney, expresses the alpha 2C-adrenergic receptor and is the prototypical cell line for the alpha 2C receptor subtype. The cloned human alpha 2C-C4 and rat RG10 receptors have been shown to express alpha 2C pharmacology. Here we report the cloning and expression of the OK alpha 2C-adrenergic receptor, OKc2. The receptor has 64% deduced amino acid identity and 21% similarity to the alpha 2-C4 receptor, giving an overall similarity of 85%. The clone, expressed in Chinese hamster ovary cells, has a pharmacology that correlates very well (r = 0.97) with that of the native OK cell alpha 2C-adrenergic receptor, and it is negatively coupled to adenylyl cyclase.

Expression of alpha 2-adrenergic receptor genes in rat tissues.

Alpha-2 adrenergic receptor subtypes are coded for by three genes. Pharmacologically alpha 2 adrenergic receptors can be classified into four subtypes: alpha 2A, alpha 2B, alpha 2C, and alpha 2D. Although pharmacologically distinct, the amino acid sequences of the alpha 2A and alpha 2D subtypes are approx 90% identical and have not been detected in a single species. Thus, they should be considered species orthologs and may be referred to as alpha 2A/D. The tissue distribution of the mRNAs for the rat alpha 2A/D, alpha 2B, and alpha 2C was analyzed utilizing RNase protection assays with probes directed to the third cytoplasmic loops. Alpha-2 adrenergic receptor mRNA was found in all tissues tested. Kidney, brain, and spinal cord had transcripts for all three subtypes. Only one mRNA subtype was detected in several tissues. Aorta and spleen had only alpha 2A/D mRNA, whereas heart and liver had only alpha 2B mRNA. All other tissues had two alpha 2 adrenergic subtype transcripts present. In contrast to the rat CNS, which contains predominantly alpha 2A/D and alpha 2C mRNA with little alpha 2B mRNA, peripheral tissues contain predominantly alpha 2A/D and alpha 2B mRNA with little alpha 2C mRNA.

The cloning and expression of an OK cell cDNA encoding a 5-hydroxytryptamine1B receptor.

Serotonin (5-hydroxytryptamine or 5-HT) is an important biogenic amine that functions as both a neurotransmitter and a hormone in the central nervous system (CNS) and the periphery. We report here the isolation of a cDNA from the OK cell that encodes a serotonin receptor (OKc1). When expressed in cultured cells, it displayed the pharmacological profile and negative coupling with adenylyl cyclase characteristic of a 5-HT1B receptor subtype. Similar to the cloned rodent 5-HT1B receptors, it had high affinity for the beta-adrenergic ligand [125I]iodocyanopindolol, because of the presence of an asparagine instead of a threonine residue in the seventh transmembrane region. The ligands used displayed the following rank order of potencies: cyanopindolol > RU24969 > methiothepin > serotonin > sumatriptan > methysergide > 8-OH-DPAT > isoproterenol. This profile correlates well (r = 0.97) with the native OK cell 5-HT1B receptor. When OKc1 is compared to the rat, mouse, and human 5-HT1B receptors, it has an amino acid sequence identity of 82%, but it is only 54% identical to the human 5-HT1D receptor.