Agonist-induced internalisation of the vasoactive intestinal polypeptide receptor (VPAC2)
To investigate agonist-induced internalisation of the VPAC<SUB>2</SUB> receptor we generated a C-terminally epitope-tagged receptor (VPAC<SUB>2</SUB>-HA) and mutant receptors with serial C-terminal truncations. Addition of the epitope tag had no significant effect on the seco...
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ndltd-bl.uk-oai-ethos.bl.uk-6495642017-08-30T03:12:36ZAgonist-induced internalisation of the vasoactive intestinal polypeptide receptor (VPAC2)Dinnis, Diane1999To investigate agonist-induced internalisation of the VPAC<SUB>2</SUB> receptor we generated a C-terminally epitope-tagged receptor (VPAC<SUB>2</SUB>-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 VPAC<SUB>2</SUB> receptor stably expressed in the human embryonic kidney cell line (HEK293). Iodinated helodermin, a VPAC<SUB>2</SUB> receptor agonist, was rapidly internalised in cells expressing the VPAC<SUB>2</SUB> and VPAC<SUB>2</SUB>-HA receptors following incubation at room temperature or at 37°C. Internalisation kinetics of the VPAC<SUB>2</SUB> and VPAC<SUB>2</SUB>-HA receptor were indistinguishable. In serum starved cells, the epitope-tagged VPAC<SUB>2</SUB> 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 VPAC<SUB>2</SUB> receptor prevents agonist-induced receptor phosphorylation. Nonetheless, truncated receptors were still able to internalise, indicating that phosphorylation is not an absolute requirement for VPAC<SUB>2</SUB> receptor internalisation. The truncated VPAC<SUB>2</SUB> receptor did not desensitise so we postulate that phosphorylation is necessary for this phenomenon. VPAC<SUB>2</SUB> receptors colocalise with transferrin receptors, which are internalised via clathrin pits and act as a marker of endosomes. No colocalisation was observed between the VPAC<SUB>2</SUB> 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 VPAC<SUB>2</SUB> receptor.615.1University of Edinburghhttp://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.649564http://hdl.handle.net/1842/22151Electronic Thesis or Dissertation |
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615.1 Dinnis, Diane Agonist-induced internalisation of the vasoactive intestinal polypeptide receptor (VPAC2) |
description |
To investigate agonist-induced internalisation of the VPAC<SUB>2</SUB> receptor we generated a C-terminally epitope-tagged receptor (VPAC<SUB>2</SUB>-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 VPAC<SUB>2</SUB> receptor stably expressed in the human embryonic kidney cell line (HEK293). Iodinated helodermin, a VPAC<SUB>2</SUB> receptor agonist, was rapidly internalised in cells expressing the VPAC<SUB>2</SUB> and VPAC<SUB>2</SUB>-HA receptors following incubation at room temperature or at 37°C. Internalisation kinetics of the VPAC<SUB>2</SUB> and VPAC<SUB>2</SUB>-HA receptor were indistinguishable. In serum starved cells, the epitope-tagged VPAC<SUB>2</SUB> 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 VPAC<SUB>2</SUB> receptor prevents agonist-induced receptor phosphorylation. Nonetheless, truncated receptors were still able to internalise, indicating that phosphorylation is not an absolute requirement for VPAC<SUB>2</SUB> receptor internalisation. The truncated VPAC<SUB>2</SUB> receptor did not desensitise so we postulate that phosphorylation is necessary for this phenomenon. VPAC<SUB>2</SUB> receptors colocalise with transferrin receptors, which are internalised via clathrin pits and act as a marker of endosomes. No colocalisation was observed between the VPAC<SUB>2</SUB> 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 VPAC<SUB>2</SUB> receptor. |
author |
Dinnis, Diane |
author_facet |
Dinnis, Diane |
author_sort |
Dinnis, Diane |
title |
Agonist-induced internalisation of the vasoactive intestinal polypeptide receptor (VPAC2) |
title_short |
Agonist-induced internalisation of the vasoactive intestinal polypeptide receptor (VPAC2) |
title_full |
Agonist-induced internalisation of the vasoactive intestinal polypeptide receptor (VPAC2) |
title_fullStr |
Agonist-induced internalisation of the vasoactive intestinal polypeptide receptor (VPAC2) |
title_full_unstemmed |
Agonist-induced internalisation of the vasoactive intestinal polypeptide receptor (VPAC2) |
title_sort |
agonist-induced internalisation of the vasoactive intestinal polypeptide receptor (vpac2) |
publisher |
University of Edinburgh |
publishDate |
1999 |
url |
http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.649564 |
work_keys_str_mv |
AT dinnisdiane agonistinducedinternalisationofthevasoactiveintestinalpolypeptidereceptorvpac2 |
_version_ |
1718520724080033792 |