Characterization of α₂-adrenergic receptor subtype-specific antibodies

Subtypes of α₂-adrenergic receptors have been defined pharmacologically in a variety of mammalian tissues. The α(2A), α(2B), α(2C), and most recently α(2D) subtypes have been characterized by their affinities for selective receptor antagonists and agonists. The genes that may encode the α(2A), α(2B), and α(2C) subtypes have been identified in human and rat. In human these genes are termed α₂-C10, α₂-C2, and α₂-C4, respectively, based on their chromosomal localization, whereas three genes, designated RG20α₂, RNGα₂, and RG10α₂, are thought to be the corresponding rat homologues. These assignments were based on the pharmacology of the cloned receptor genes expressed in transfected cells and on the detection of homologous mRNAs by Northern blot analyses in cell lines or tissues with pharmacologically defined α₂-adrenergic receptors. However, the subtype assignment of cloned genes has not been fully resolved by these means. To help clarify the subtype assignment, we have raised antibodies against sequences from the divergent third intracellular loop of the human and rat α₂-adrenergic receptors. These antibodies were found to be subtype specific in immunoprecipitating either the cloned receptors expressed by DNA transfection or the pharmacologically defined receptors prepared from various tissues. Our immunological data corroborate the assignments of α₂- C2/RNGα₂ as encoding the α(2B) subtype in NG108-15 cells and rat neonatal lung and of α₂-C4/RG10α₂ as encoding the α(2C) subtype in opossum kidney cells. Furthermore, antibodies against α₂-C10 and RG20α₂ but not α₂- C2/RNGα₂ or α₂-C4/RG10α₂ were both found to recognize α₂-adrenergic receptors expressed in rat submaxillary glands and in bovine pineal gland, two tissues with α(2D) pharmacology. Because three genes were identified in the rat and human genome, these data suggest that the pharmacologically defined 'α(2D) receptor' is genetically of the α(2A) subtype.