Markovic, Danijela (2007) Investigation of the corticotropin-releasing hormone receptor signalling properties. PhD thesis, University of Warwick.

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Abstract

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 (PhD)
Subjects:	Q Science > QP Physiology
Library of Congress Subject Headings (LCSH):	Corticotropin releasing hormone -- Receptors
Official Date:	July 2007
Dates:	Date Event July 2007 Submitted
Institution:	University of Warwick
Theses Department:	Warwick Medical School
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Grammatopoulos, Dimitris ; Lehnert, Hendrik
Extent:	xix, 318 leaves
Language:	eng

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