Characterization of Serum and Glucocorticoid Kinase Isoforms and a Membrane Glucocorticoid Receptor in the Euryhaline Tilapia (Oreochromis mossambicus): Implications in Salinity Adaptation.

JOHNSTONE III, WILLIAM MILLER. Characterization of Serum and Glucocorticoid Kinase Isoforms and a Membrane Glucocorticoid Receptor in the Euryhaline Tilapia (Oreochromis mossambicus): Implications in Salinity Adaptation. (Under the direction of Russell J. Borski). Euryhaline fishes, such as the Mozambique tilapia (Oreochromis mossambicus) have the capacity to regulate plasma osmolalility under salinities ranging from freshwater (FW) to salinities in excess of full-strength seawater (SW). This highly orchestrated adaptation process is regulated by multiple endocrine factors in teleosts including prolactin, which is implicated in freshwater (FW) adaptation and cortisol, the main glucocorticoid (GC) and mineralcorticoid (MC) in teleosts, which is implicated in SW and FW adaptation. These hormones act directly by regulating ion transport machinery in osmoregulatory epithelialia such as the gill, kidney and intestine and indirectly through organs that secrete osmoregulatory hormones. Modulation of ion pumps and effects on hormone secretion require functional receptors at the target tissue as well as intracellular signaling proteins such as kinases. Moreover, research suggests that signal transduction pathways involving GCs in vertebrates not only operate through a classical, genomic mechanism, but also via a rapid, nongenomic mechanism mediated by a plasma membrane binding site. The aim of this research was threefold: 1) clone and characterize a signaling kinase, serum and glucocorticoid-induced kinase (SGK), that plays a role in ion transport in mammals and amphibians but remains uncharacterized in teleosts; 2) evaluate regulation of SGK isoforms during chronic and acute hypoand hyperosmotic stress; 3) and identify and pharmacologically characterize a membrane glucocorticoid (cortisol) receptor (mGR) in the Mozambique tilapia that may transduce rapid responses of cortisol in regulating ion transport and secretion of osmoregulatory hormones. We found three isoforms of SGK (SGK1, -2, and -3) in the gill of Mozambique tilapia that share a high degree of similiarity with SGK isoform sequences from other vertebrates at the nucleotide and amino acid level. Examination of the predicted tertiary structure of these proteins demonstrated a high degree of structural homology as well. Subsequent analysis of the distribution of the SGK isoforms revealed that SGK1 and SGK3 were expressed ubiquitously across the tissues examined, although SGK1 was expressed in higher amounts. SGK2, however, demonstrated a much more specific tissue distribution as it was found in much higher amounts in the kidney than any other tissue and was virtually absent from the brain and pituitary. When expression profiles of gill SGK isoforms 1 and 2 were generated, a differential pattern of regulation of SGK isoforms with regard to salinity was revealed. SGK1 is regulated by hypoand hyperosmotic environments and is upregulated after 24 hours in SW and after 0.5 and 24 hours in FW, suggesting an acute and chronic regulatory role for SGK1 in osmoregulation. Gill SGK2 mRNA, however, is upregulated in FW at 24 hours, whereas it declines in SW, suggesting a hyperosmoregulatory role for this isoform. Binding studies in Mozambique tilapia revealed a high affinity, low capacity mGR in the liver and a lower affinity, low capacity mGR in the kidney. The liver mGR displays a pharmacological signature distinct from the intracellular glucorticoid receptor (iGR), demonstrating no affinity for an iGR agonist (dexamethasone) and antagonist (RU486). Association and dissociation binding kinetics also were atypical of an intracellular GR, but are more comparable to membrane receptors. The data presented here characterize, for the first time, a novel mGR and three isoforms of SGK in teleosts, both of which may be involved in regulating genomic and nongenomic pathways facilitating salinity adaptation in euryhaline fishes. The reported nongenomic actions of GC on osmoregulatory organs in this species may involve signal transduction pathways involving the mGR and multiple isoforms of SGK. Characterization of Serum and Glucocorticoid Kinase Isoforms and a Membrane Glucocorticoid Receptor in the Euryhaline Tilapia (Oreochromis mossambicus): Implications in Salinity Adaptation by William Miller Johnstone III A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy