Investigation of the corticotropin-releasing hormone receptor signalling properties
Markovic, Danijela (2007) Investigation of the corticotropin-releasing hormone receptor signalling properties. PhD thesis, University of Warwick.
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Beside the well-known role of CRH and CRH-related peptides in controlling HPA axis, the peptides have been implicated as important mediators various physiological processes including reproduction, endocrinology of pregnancy, energy homeostasis. Diverse functions of CRH and UCNs are governed via activation of two types of CRH receptors, R1 and R2. Since tissue sensitivity to agonists is determined by the availability of receptor in the plasma membrane and how fast the signal is terminated, one of goals of this project was to investigate the cellular expression of CRH-R1 variants (α, β, d and β/d), and internalization characteristics of the receptors following homologous and heterologous activation of the receptor. Also, I investigated the structural and functional characteristics of the CRH-R1d receptor, a splice variant that contains deletion of 14 amino acids within the 7th TMD. The co-expression studies utilizing HEK293 cells expressing CRH-R1d and CRH-R2β demonstrated attenuation of CRH-R2β mediated cAMP production and MAPK activation in the presence of CRH-R1d. This could be of potential importance in human peripheral tissues which express both types of CRH receptors, such is uterus. Additionally, I investigated the signalling and internalization characteristics of CRH-R2β and explore the possible link between the CRH-R2 internalization and MAPK activation. The analysis of the spatio-temporal characteristics of MAPK activation revealed important differences between CRH-R1 and R2 mediated signalling cascades. Immunofluorescence analysis demonstrated that activation of both type of CRH receptors led to a recruitment of β-arrestin to the plasma membrane; however the internalization pathways of the receptors were different. Since human pregnancy is associated with changes in the myometrial CRH-R variant expression profile and functional activity, as a part of the study, I characterised the effect of IL-1β (an important mediator of the onset of labour) on the regulation of CRHR1 gene expression and the functional properties of the CRH-R. Data showed that IL-1β can potentially target CRH-R1 gene transcription and splicing mechanisms of the CRHR1 gene; these interactions appeared to involve two members of the MAPK family of proteins, ERK1/2 and p38 MAPK and NF-κB activation. Interestingly, increased CRHR1 gene transcription and generation of receptor splice variants was not associated with increased CRH-R protein levels and CRH signalling activity. Furthermore, the signalling characteristics of CRH-R activated by UCN-II (CRH-R2 specific agonist) were investigated. The data showed that activation of CRH-R2 did not lead to cAMP production which is associated with the quiescent state of uterus, but the MAPK signalling cascade was activated, which have been implicated in mediating pathways that promote contractility. During the course of this project biological role of CRH receptors was investigated in T37i cells. RT-PCR analysis showed the presence of CRH-R1 and R2 mRNA in mice brown adipose tissue and T37i cells. Immunofluorescence and western blot analysis demonstrated the presence of CRH-Rs in T37i cells. The functional capacity of adipose CRH-Rs to activate adenylyl cyclase and MAPK signalling cascade was assessed. Low concentration of agonists (close to receptors Kd=1 nM) stimulated activation of adenylyl cyclase/cAMP/PKA signalling cascade resulting in lipolysis. However; higher concentration (10-10 nM) of agonists activated MAPK signalling cascades.
|Item Type:||Thesis or Dissertation (PhD)|
|Subjects:||Q Science > QP Physiology|
|Library of Congress Subject Headings (LCSH):||Corticotropin releasing hormone -- Receptors|
|Institution:||University of Warwick|
|Theses Department:||Warwick Medical School|
|Supervisor(s)/Advisor:||Grammatopoulos, Dimitris ; Lehnert, Hendrik|
|Extent:||xix, 318 leaves|
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