Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.478849
Title: Molecular cloning and pharmacological characterisation of the rat CRF receptor type 2 beta and identification of a novel splice variant
Author: Rainard, Julie M.
ISNI:       0000 0001 3505 5587
Awarding Body: Nottingham Trent University
Current Institution: Nottingham Trent University
Date of Award: 2008
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Abstract:
Corticotropin-releasing factor (CRF) receptors are G-protein coupled receptors that play an important role in the body's response to stressors and help maintain homeostasis. They are involved in numerous pathologies including anxiety, depression and eating disorders. In this study, the 5'-end unique to the rat CRF receptor type 2 beta (CRF-R2β) cDNA was cloned and the full-length construct was generated. This was transfected into mammalian Chinese Hamster Ovary (CHO) cells. The pharmacological characterisation of the rat CRF-R2β, particularly with respect to different natural mammalian agonists, e.g. CRF, urocortin I, II and III, was achieved by measuring intracellular cAMP levels (using a non-radioactive cAMP high sensitivity luminescence assay), since the CRF-R2 couples to Gas proteins, thus activates the adenylyl cyclase pathway. Urocortin I was the most potent agonist at the rat CRF-R2β, followed by the equipotent urocortin II and III, and CRF was the least potent agonist. The selective CRF-R2 antagonist, antisauvagineSO was shown to inhibit CRF-induced cAMP accumulation in a concentration-dependent manner and presented high affinity for the rat CRF-R2β. Inhibitors of the Gai and Gaq pathways were also tested, and although the rat CRF-R2β did not appear to couple to Gai proteins, signalling via the Gaq/inositol triphosphate pathway could not be excluded. A novel splice variant of the CRF-R2 gene was discovered during the cloning experiments. This alternatively-spliced form of the receptor lacks 21 amino acids, which are encoded by the ninth exon of the CRF-R2 gene (corresponding to part of the fourth transmembrane domain and the second extracellular loop of the receptor). The mRNAs for this new isoform and the full-length CRF-R2β were shown to co-localise in various tissues from both neonatal and adult rats. A similar result was found in human tissues suggesting evolutionary conservation of this splice variant among mammalian species. Western blot analysis using an anti-CRF-R2 antibody confirmed that the truncated receptor protein was synthesised in CHO cells, however, the missing sequence was expected to affect the receptor topology. Indeed, the possible re-arrangement of the polypeptide chain into a protein with five transmembrane domains and with a longer first extracellular loop was predicted. Although the truncated receptor isoform failed to exhibit a pharmacological response when expressed on its own, a reduction in the potency of the four mammalian agonists was observed, when the two receptor cDNAs were co-transfected in CHO cells. This indicated a potential role for this new splice form in modulating the wild-type CRF-R2β function, possibly via dimerisation. Finally, the regulation of CRF-R2 gene expression under nutrient stress was investigated using the rat smooth muscle A7r5 cell line. Serum deprivation induced the down-regulation of the CRF-R2 gene, as demonstrated by a decreased pharmacological response and a reduction in CRF-R2β mRNA and protein levels. Therefore, the CRF-R2 gene could be a candidate for downregulation by the serum response factor transcription factor.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.478849  DOI: Not available
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