Calcitonin gene-related peptide (CGRP) and adrenomedullin (ADM) receptors are heterodimeric complexes composed of the calcitonin receptor-like receptor (CLR) and a receptor activity-modifying protein (RAMP). Association with RAMP1 gives a high affinity CGRP receptor, whereas association with RAMP2 or RAMP3 gives high affinity ADM receptors. CGRP and ADM are widely distributed throughout 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) production and inducible NO synthase (iNOS) expression in trigeminal ganglion glial cells via ERK activation. CGRP is known to induce iNOS/NO production in thoracic artery smooth muscle cells (TA-SMC) pretreated with interleukin-1b. However, the molecular mechanism of CGRP-induced iNOS/NO production in TA-SMC is unknown. Therefore, in order to determine if CGRP induces iNOS/NO production via ERK activation, I first investigated the exact mechanisms through which CGRP activates 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 pathway responsible for iNOS induction. Compared to CLR•RAMP1 and CLR•RAMP3, little is known about the postendocytic sorting of CLR•RAMP2. Using HEK cells stably expressing CLR•RAMP2, I investigated the molecular mechanisms regulating the ADM receptor. I first showed that, unlike CLR•RAMP1, even transient stimulation of CLR•RAMP2 with ADM promotes degradation of both CLR and RAMP2, indicating that this ADM receptor does not recycle to the cell-surface. Moreover, I showed that CLR, not RAMP2, is constitutively ubiquitinated, which was further enhanced upon ADM stimulation. In order to elucidate the role of ADM-mediated ubiquitination of CLR, I made a lysine-less mutant of CLR, named CLRD9KR. I showed that ubiquitination of CLR did not affect ADM-induced trafficking of CLR•RAMP2 to lysosomes, nor did it affect the degradation or the ERK signalling of CLR•RAMP2. However, I showed that ubiquitination of CLR regulated the rate of degradation of the receptor. Together, these results indicate that CLR•RAMP2 does not recycle and is degraded via a molecular mechanism that is accelerated by ADM-induced ubiquitination of CLR.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:604647 |
| Date | January 2013 |
| Creators | Roux, Benoit Thomas |
| Contributors | Cottrell, Graeme ; Ward, Stephen |
| Publisher | University of Bath |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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