Guanine-Nucleotide Binding Proteins, Tyrosine Kinases, and Associated Regulators in the Integrated Control of Basal and GnRH-Dependent Goldfish Pituitary Hormone Secretion
Creators: Khalid, Enezi
Publisher: name: University of Alberta Library
Year: 2023

<p>In vertebrates, the pituitary gland is a major regulator of physiological processes through the production and release of chemical signal messengers called hormones. In turn, central control of pituitary cell functions is exerted through neurohormones secreted from hypothalamic neurons. The fidelity of hormonal communication is achieved via recognition molecules, termed receptors, which elicit intracellular responses when specifically bound and activated by cognate hormone ligands. Among these receptors is the family of guanine-nucleotide (G) protein-coupled receptors (GPCRs), which are the predominant class of cell-surface transmembrane recognition systems for a variety of hormones and neuropeptides.<br>
Among pituitary hormones, luteinizing hormone (LH) controls processes of sexual maturation and reproduction, whereas growth hormone (GH) influences somatic growth, metabolism, and immune function. In the goldfish (Carassius auratus), the two natural variants of hypothalamic gonadotropin-releasing hormone, GnRH2 and GnRH3, both stimulate LH and GH release by activating a shared population of plasma membrane G protein-coupled GnRH receptors (GnRHRs) on pituitary gonadotrophs and somatotrophs (LH- and GH- secreting cells, respectively). Prior research in our lab has characterized multiple post-receptor signal transduction mechanisms mediating the goldfish pituitary hormone release responses to GnRH. In addition to the shared use of several conserved intracellular signalling modules, GnRH-isoform-, cell-type-, and function-selective engagement of signalling effectors also exists in this system. Although ligand-selective recruitment of effectors following GnRHR activation is established, the functions of proximal receptor elements in driving such “biased signalling” remains unresolved. <br>
In GPCR systems across taxa, the classical immediate effectors including heterotrimeric guanine nucleotide-binding (G) proteins, β-arrestins, and G protein-coupled receptor kinases (GRKs) play agonist-specific roles in facilitating both GPCR signal transduction and signal termination. Additionally, monomeric “small GTPases” are another relevant class of intracellular effectors in secretory cell types, due to their highly conserved cellular functions in modulating vesicle exocytosis via dynamic control of the actin cytoskeleton. However, the roles of these effectors in neuroendocrine systems and GPCR networks, especially in basal vertebrate models, have been under studied. Likewise, GPCR engagement of intracellular protein tyrosine kinases and other transmembrane receptor systems through transactivation may represent an important non-canonical route of directing selective cell functions. Altogether, these effectors represent important nodes of signal transduction that may be engaged by GnRHRs in ligand-selective fashions to control hormone secretion from goldfish pituitary cells.<br>
The aims of my thesis were to characterize the involvement of classical GPCR effectors, small GTPases, and non-canonical tyrosine kinase elements in the control of goldfish pituitary cell secretion in basal (unstimulated) states and in response to GnRH isoforms. To achieve these aims, I utilized the established dispersed pituitary cell column perifusion system to monitor LH and GH secretion responses following transient pharmacological manipulation of the effectors of interest, in basal and GnRH-stimulated conditions. Results show that several classical GPCR-interacting proteins play important roles in mediating secretion responses to GnRH. In particular, multiple G protein alpha subtypes control GnRH-dependent effects, while β-arrestins and GRKs are identified for the first time as novel effectors involved in both mediating and terminating hormone release responses to GnRH, in a ligand- and pituitary-cell-type-dependent manner. In addition, studies of small GTPase proteins reveal how these cellular effectors can be differentially utilized in basal and agonist-stimulated cell states to control hormone exocytosis. Furthermore, intracellular protein tyrosine kinases, receptor tyrosine kinases, and additional non-canonical cell-surface elements are identified for the first time in mediating selective GnRH-dependent and basal effects in this system.<br>
Overall, results from my thesis expand the known repertoire of GnRH signalling networks across taxa and highlight the diversity in transduction machineries utilized to evoke cellular responses in response to extracellular signals. Findings also reveal, and lay the foundation for understanding, how the selective usage of intracellular effectors can be leveraged during the coordinated control of reproductive and growth processes by hypothalamic neuropeptide-GPCR systems. Just as importantly, this thesis represents one of only a handful of detailed investigations into natural ligand bias in physiological study systems, especially from any basal vertebrate model.</p>