Agonist-induced internalization and desensitization of the apelin receptor

George R. Pope, Sharada Tilve, Craig A. McArdle, Stephen J. Lolait, Anne-Marie O'Carroll

Research output: Contribution to journalArticle (Academic Journal)peer-review

11 Citations (Scopus)
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Abstract

Apelin acts via the G protein-coupled apelin receptor (APJ) to mediate effects on cardiovascular and fluid homeostasis. G protein-coupled receptor (GPCR) trafficking has an important role in the regulation of receptor signalling pathways and cellular functions, however in the case of APJ the mechanisms and proteins involved in apelin-induced trafficking are not well understood. We generated a stable HEK-293 cell line expressing N-terminus HA-tagged mouse (m) APJ, and used a semi-automated imaging protocol to quantitate APJ trafficking and ERK1/2 activation following stimulation with [Pyr1]apelin-13. The mechanisms of [Pyr1]apelin-13-induced internalization and desensitization were explored using dominant-negative mutant (DNM) cDNA constructs of G protein-coupled receptor kinase 2 (GRK2), β-arrestin1, EPS15 and dynamin. The di-phosphorylated ERK1/2 (ppERK1/2) response to [Pyr1]apelin-13 desensitized during sustained stimulation, due to upstream APJ-specific adaptive changes. Furthermore, [Pyr1]apelin-13 stimulation caused internalization of mAPJ via clathrin coated vesicles (CCVs) and also caused a rapid reduction in cell surface and whole cell HA-mAPJ. Our data suggest that upon continuous agonist exposure GRK2-mediated phosphorylation targets APJ to CCVs that are internalized from the cell surface in a β-arrestin1-independent, EPS15- and dynamin-dependent manner. Internalization does not appear to contribute to the desensitization of APJ-mediated ppERK1/2 activation in these cells.
Original languageEnglish
Pages (from-to)108-119
Number of pages12
JournalMolecular and Cellular Endocrinology
Volume437
Early online date1 Aug 2016
DOIs
Publication statusPublished - 5 Dec 2016

Keywords

  • G protein-coupled receptor
  • Apelin
  • Apelin receptor
  • Intracellular trafficking
  • Signaling
  • Extracellular-signal-regulated kinase (ERK)

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