Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.649564
Title: Agonist-induced internalisation of the vasoactive intestinal polypeptide receptor (VPAC2)
Author: Dinnis, Diane
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1999
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Abstract:
To investigate agonist-induced internalisation of the VPAC2 receptor we generated a C-terminally epitope-tagged receptor (VPAC2-HA) and mutant receptors with serial C-terminal truncations. Addition of the epitope tag had no significant effect on the second messenger signalling or agonist binding properties of the VPAC2 receptor stably expressed in the human embryonic kidney cell line (HEK293). Iodinated helodermin, a VPAC2 receptor agonist, was rapidly internalised in cells expressing the VPAC2 and VPAC2-HA receptors following incubation at room temperature or at 37°C. Internalisation kinetics of the VPAC2 and VPAC2-HA receptor were indistinguishable. In serum starved cells, the epitope-tagged VPAC2 receptor was predominantly located in the plasma membrane. Treatment with VIP caused a marked shift in receptor distribution from the plasma membrane to a single intracellular site. Receptor internationalisation was dependent upon the concentration of agonist, incubation time and temperature. Removal of agonist resulted in the reappearance of receptor immunoreactivity at the plasma membrane; this movement was unaffected by the presence of the protein synthesis inhibitor cycloheximide, suggesting that the majority of receptors are recycled. Other work in our laboratory has demonstrated that truncation of the C-terminal intracellular domain of the VPAC2 receptor prevents agonist-induced receptor phosphorylation. Nonetheless, truncated receptors were still able to internalise, indicating that phosphorylation is not an absolute requirement for VPAC2 receptor internalisation. The truncated VPAC2 receptor did not desensitise so we postulate that phosphorylation is necessary for this phenomenon. VPAC2 receptors colocalise with transferrin receptors, which are internalised via clathrin pits and act as a marker of endosomes. No colocalisation was observed between the VPAC2 receptor and a marker for the trans-golgi network, indicating that the receptor does not recycle through this compartment. This work provides the first direct evidence for agonist-induced internalisation of the VPAC2 receptor.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.649564  DOI: Not available
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