Characterisation of the Molecular Mechanisms Regulating the Signalling and Post-endocytic sorting of the Receptors for Calcitonin Gene-Related Peptide and Adsenomedullin

Abstract

Calcitonin gene-related peptide (CGRP) and adrenomedullin (ADM)receptors are heterodimeric complexes composed of the calcitonin receptor-likereceptor (CLR) and a receptor activity-modifying protein (RAMP). Association withRAMP1 gives a high affinity CGRP receptor, whereas association with RAMP2 orRAMP3 gives high affinity ADM receptors. CGRP and ADM are widely distributedthroughout the body and play important roles and are implicated in many diseases including migraine, heart failure and sepsis.Recently, CGRP has been shown to promote nitric oxide (NO) productionand inducible NO synthase (iNOS) expression in trigeminal ganglion glial cells viaERK activation. CGRP is known to induce iNOS/NO production in thoracic arterysmooth muscle cells (TA-SMC) pretreated with interleukin-1b. However, themolecular mechanism of CGRP-induced iNOS/NO production in TA-SMC isunknown. Therefore, in order to determine if CGRP induces iNOS/NO productionvia ERK activation, I first investigated the exact mechanisms through which CGRPactivates ERK1-2 in HEK cells. By using different inhibitors I showed that CGRPinduced ERK activation is mainly activated through two major pathways. I showed for the first time that CGRP induces ERK activation through transactivation of ErbB1 and as expected through the cAMP/PKA pathway. Then, in order to characterise a suitable model to study CGRP-induced iNOS expression, I used primary TA-SMC and I showed that CGRP induces iNOS upregulation, which is reduced when cells are incubated with U0126, a MEK inhibitor. Thus, these results suggest that CGRP induces iNOS expression via ERK activation in TA-SMC.However, further experimentation is required to determine the exact ERK pathwayresponsible for iNOS induction.Compared to CLR•RAMP1 and CLR•RAMP3, little is known about the postendocytic sorting of CLR•RAMP2. Using HEK cells stably expressingCLR•RAMP2, I investigated the molecular mechanisms regulating the ADMreceptor. I first showed that, unlike CLR•RAMP1, even transient stimulation ofCLR•RAMP2 with ADM promotes degradation of both CLR and RAMP2, indicatingthat this ADM receptor does not recycle to the cell-surface. Moreover, I showedthat CLR, not RAMP2, is constitutively ubiquitinated, which was further enhancedupon ADM stimulation. In order to elucidate the role of ADM-mediatedubiquitination of CLR, I made a lysine-less mutant of CLR, named CLRD9KR. Ishowed that ubiquitination of CLR did not affect ADM-induced trafficking ofCLR•RAMP2 to lysosomes, nor did it affect the degradation or the ERK signallingof CLR•RAMP2. However, I showed that ubiquitination of CLR regulated the rateof degradation of the receptor. Together, these results indicate that CLR•RAMP2does not recycle and is degraded via a molecular mechanism that is acceleratedby ADM-induced ubiquitination of CLR.

Date of Award	29 Jul 2013
Original language	English
Awarding Institution	University of Bath
Supervisor	Graeme Cottrell (Supervisor) & Stephen Ward (Supervisor)