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Title: Modulation of neuropeptide growth factor signalling by anti-cancer substance P analogues
Author: Tufail-Hanif, U.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2005
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Model cell systems consisting of CHO-K1 cell-lines stably expressing GRP or V1A receptors were established and the effect of SP-D and SP-G tested. Expression of GRP and V1A receptors led to the development of a transformed phenotype as cells showed increased cloning efficiency and survival in soft-agar and suspension growth respectively. GRP and V1A receptor expressing cells were less adherent, more migratory and not contact inhibited. Neuropeptide receptor stimulation provided some protection from the cytotoxic effects of etoposide suggesting a role in chemoresistance. Substance-P analogues inhibited normal and anchorage-independent growth of receptor expressing cells. In receptor binding studies on GRP and V1A receptor expressing cells, analogue inhibited radioligand binding non-competitively. Transfected GRP and V1A receptors effectively coupled to Gαq to increase intercellular calcium and the analogues were effective antagonists of this response. Neuropeptide and analogues stimulated ERK activity in GRP and V1A receptor expressing cells. Activation of ERK by neuropeptide was rapid and transient while analogue induced activation was delayed and sustained. Analogue-stimulated ERK activity was pertussis toxin sensitive whereas neuropeptide-stimulated ERK activation was not. In addition, analogue induced ERK activity was blocked by inhibition of EGF receptor kinase. This indicates that SP-D and SP-G facilitate receptor coupling to G-protein Gi/Go subunits for subsequent calcium-independent ERK activation via EGFR transactivation. Stable cell-lines expressing different levels of V1A receptor were used to examine the effect of altering the ratio of receptor to G-protein on the ability of the analogues to direct receptor signalling. Chimeric V1A receptors containing the second (V1i2) on third intracellular (V1i3) loop of the V2 receptor were used to investigate the influence of substance-P analogues on G-protein selectivity. Both receptors were still capable of binding AVP and Sp-G but had altered ability to activate PLC and ERK. The second intracellular loop of V1AR was essential for AVP-stimulated PLC and ERK activation but not for SP-G-induced ERK activation. This confirms that the effects of the agents cause an alteration in the receptor-G-protein coupling domains of receptors. These findings demonstrate that substance-P analogues are biased agonists of receptors other than GRP receptors, activating downstream signals which differ fro those stimulated by the natural agonist through promoting an alternative agonist state of the receptor. This pathway selectivity combined with the receptor specificity of different substance-P analogues offers great potential for the tailored treatment of neuropeptide-dependent tumours.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available