Localization of alpha-2 adrenergic receptor subtypes using subtype-specific antibodies.

Abstract

Three subtypes of alpha-2 adrenergic receptors (α₂AR) have been identified: α₂A, α₂B and α₂C. α₂ARs are known to mediate a number of functions in many tissues. It is not clear, however, what the distribution of α₂AR subtypes is in these tissues. The distribution of α₂AR subtypes is fundamental to understanding receptor function and the development of more effective and specific α₂-adrenergic agents for therapeutic use. The working hypothesis for this dissertation is that there are specific subtypes of α₂ARs in tissues and in cells that have discrete localizations and may subserve different physiological function. To test this hypothesis two specific aims have been raised. The first aim was: the generation of subtype-specific antibodies. Experiments were conducted to express fusion proteins containing part of the third intracellular loop of each α₂AR. Chickens were immunized with fusion proteins and antibodies were purified from the yolk of the chicken eggs. The antibodies have complete subtype specificity as characterized by immunofluorescence studies on COS-7 cells transfected with DNA encoding the α₂ARs. The second aim was: the immunohistochemical localization of α₂AR subtypes in the primary culture of rat spinal cord, anterior segments of human and rabbit eyes and transfected COS-7 cells. Experiments were conducted to localize the α₂AR subtypes in cultured cells and tissues using indirect immunofluorescence techniques. The immunofluorescence results were confirmed by alternative approaches, e.g. reverse transcription-PCR, ligand binding and cAMP assay. The experimental results showed that multiple α₂AR subtypes are expressed in one tissue and that specific subtypes of α₂ARs are expressed in different tissues. Using a dual-labeling technique, two subtypes were co-localized in the same neuronal cell of the rat spinal cord. Furthermore, the immunofluorescence studies on the transfected COS-7 cells showed that the three subtypes of α₂ARs displayed different subcellular localization. Taken together the results of the studies presented in this dissertation provide evidence in support of the working hypothesis. The present work has provided a new opening in the study of the localization of α₂AR subtypes. This information provides new insights into the understanding of α₂AR functions in tissues.