Regulation of the orexin 1 receptor by beta-arrestins

Milasta, Sandra (2004) Regulation of the orexin 1 receptor by beta-arrestins. PhD thesis, University of Glasgow.

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The orexin 1 receptor was identified as an orphan G protein-coupled receptor (GPCR) in 1998 (Sakurai et ah, 1998). There is great interest in the orexin receptor system since it is involved in the control of feeding and energy metabolism (Sakurai et al, 1998), the modulation of neuroendocrine function (van den Pol et al, 1998; Smart, 1999) and the regulation of the sleep -wake cycle (Smart, 1999). However not much is known about the regulation of the orexin 1 receptor following stimulation. beta-arrestins bind agonist- activated, phosphorylated GPCRs and mediate their desensitisation and internalisation. They may also function as GPCR signal transducers. The aim of this thesis was to investigate internalization and signalling of the orexin 1 receptor and the involvement of beta- arrestins in these processes. In HEK293T cells expressing wild type orexin 1 receptor, orexin A stimulation triggered beta-arrestin 2 binding to the receptor and co-internalisation of receptor beta-arrestin complexes via clathrin-coated vesicles into acidic endosomes, in a dynamin-dependent manner. Moreover, studies of receptor internalisation in wild type, beta-arrestin-, Src family kinase-or Gq/n-deficient mouse embryo fibroblasts revealed sequestration of the orexin 1 receptor to be beta-arrestin-dependent, but G protein-and Src-independent. Mutational analysis of the orexin 1 receptor demonstrated that high affinity binding between the receptor and beta-arrestin 2 was conferred by a single cluster of Ser/Thr residues at the extreme C-terminus. Although this mutant form of the receptor was no longer able to co-intemalise with beta -arrestin 2, the pathway and time course of receptor internalisation was unaltered. In CHO cells, orexin A challenge induced rapid receptor phosphorylation which was partly mediated by protein kinase A (PKA) and protein kinase C (PKC). Surprisingly the levels of phosphorylation were similar for the cluster Cl mutant indicating the principal phosphorylation site to be distinct from the cluster of Ser/Thr residues essential for agonist-induced recruitment of beta-arrestins. To investigate the signalling pathways elicited by addition of orexin A, mutant forms of the orexin 1 receptor unable to stimulate G protein signalling were generated. Activation of the orexin 1 receptor caused an increase in ERKl/2 activity by a process depending on an intact endocytic pathway since inhibition of endocytosis by concanavalin A or dominant negative dynamin resulted in attenuated ERKl/2 phosphorylation. However hyperosmolar levels of sucrose had no effect on ERKl/2 activation. In addition, orexin A challenge of Xiv Src family kinase knock out MEF cells expressing the orexin 1 receptor resulted in ERKl/2 stimulation. There was significant difference in the time course of ERKl/2 phosphorylation upon stimulation of the wild type receptor and the cluster Cl mutant. On the other hand, no increase in ERKl/2 phosphorylation could be observed for the mutants unable to activate G proteins. Agonist challenge of the wild type receptor also caused stimulation of the JNK MAPK pathway. In contrast to the ERKl/2 MAPK pathway stimulation of all mutants tested resulted in increased JNK activity. p38 another member of the MAPK family was not activated after agonist challenge of the orexin 1 receptor excluding an involvement of this MAPK in orexin 1 receptor signalling. Taken together these results show that a single cluster of hydroxy amino acids within the C-terminus of the orexin 1 receptor determines the affinity of the interaction with beta- arrestin 2. They also indicate a key role of beta-arrestin scaffolding in fine tuning the kinetics of orexin 1 receptor-mediated, G protein-dependent ERKl/2 activation.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Neurosciences, physiology, endocrinology.
Colleges/Schools: College of Medical Veterinary and Life Sciences
Supervisor's Name: Milligan, Prof. Graeme
Date of Award: 2004
Depositing User: Enlighten Team
Unique ID: glathesis:2004-71143
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 May 2019 10:49
Last Modified: 15 Jun 2021 10:43

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