Molecular Cloning and Functional Characterization of Goldfish Alpha-2 Adrenergic Receptors

This dissertation, "Molecular Cloning and Functional Characterization of Goldfish Alpha-2 Adrenergic Receptors" by Hoi-yan, Chan, 陳凱恩, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled MOLECULAR CLONING AND FUNCTIONAL CHARACTERIZATION OF GOLDFISH ALPHA-2 ADRENERGIC RECEPTORS Submitted by Chan Hoi Yan for the degree of Master of Philosophy at The University of Hong Kong in September 2004 In mammals, adrenergic stimulation at the hypothalamic level through alpha-2 adrenergic receptors can induce growth hormone secretion from the pituitary. However, the alpha-2 receptor subtype(s) responsible for this stimulatory action has not been elucidated. In the goldfish, we have previously shown that alpha-2 activation at the pituitary level can suppress growth hormone release and that removal of alpha-2 inhibition can trigger a rebound of growth hormone secretion. These findings have prompted us to speculate that alpha-2 adrenergic receptors should be expressed in goldfish pituitary cells and be responsible for the growth hormone regulation by adrenergic input at the pituitary level. As a first step to study the alpha-2 receptor subtype(s) expressed in the goldfish pituitary, molecular cloning was performed to establish the structural identity of the goldfish alpha-2A, alpha-2B, and alpha-2C adrenergic receptors. By library screening and 3'/5' RACE, the complementary DNAs (cDNAs) for alpha-2A, alpha-2B, and alpha-2C adrenergic receptors with the sizes of 2.72 Kb, 2.86 Kb, and 1.72 Kb, respectively, have been isolated from the brain-pituitary axis of the goldfish. The open-reading frames of these cDNAs encode proteins of 383 to 505 amino acids in size with seven transmembrane domains typical of those of the G protein-coupled receptors. Sequence analysis also reveals that these goldfish alpha-2A, alpha-2B, and alpha-2C adrenergic receptors are structurally homologous to the corresponding receptor subtypes in mammals. By reverse transcription polymerase chain reaction (RT-PCR), the transcripts of alpha-2A and alpha-2B adrenergic receptors were identified in many tissues in the goldfish, including the heart, liver, gill, intestine, kidney, muscle, spleen, pituitary, and various brain segments. A similar expression pattern was also noted for alpha-2C adrenergic receptor, except that its mRNA was not detected in the intestine and its expression in the brain was restricted to the brain stem and spinal cord. To characterize the pharmacological and biochemical properties of these newly cloned receptors, functional expression of goldfish alpha-2A, alpha-2B, and alpha-2C adrenergic receptors was conducted in Chinese hamster ovary (CHO) cells. In this case, the receptor binding properties of these goldfish adrenergic receptors were tested using a radioreceptor binding approach. Although the order of affinity of the respective alpha-2A, alpha-2B, and alpha-2C adrenergic receptors is different from that reported in mammals, these receptors consistently exhibited a high affinity for ligands selective for alpha-2 but not for alpha-1 and beta adrenergic receptors. Furthermore, activation of these receptors by norepinephrine or the alpha-2 agonist + + clonidine could trigger rapid activation of the Na /H exchanger, an inhibition of 2+ cyclic AMP production and a decrease in intracellular Ca levels. These results, taken together, have confirmed that these newly cloned receptors are functional alpha-2 adrenergic receptors in the goldfish. The cDNAs for the goldfish alpha-2A, alpha-2B, and alpha-2C