title: Agonist-induced internalization and desensitization of the apelin receptor.
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.
authors: Laboratories for Integrative Neuroscience and Endocrinology, School of Clinical Sciences, University of Bristol, Whitson Street, Bristol BS1 3NY, UK.
authors: George R
authors: GR
authors: Pope
authors: Laboratories for Integrative Neuroscience and Endocrinology, School of Clinical Sciences, University of Bristol, Whitson Street, Bristol BS1 3NY, UK.
authors: Sharada
authors: S
authors: Tilve
authors: Laboratories for Integrative Neuroscience and Endocrinology, School of Clinical Sciences, University of Bristol, Whitson Street, Bristol BS1 3NY, UK.
authors: Craig A
authors: CA
authors: McArdle
authors: Laboratories for Integrative Neuroscience and Endocrinology, School of Clinical Sciences, University of Bristol, Whitson Street, Bristol BS1 3NY, UK.
authors: Stephen J
authors: SJ
authors: Lolait
authors: Laboratories for Integrative Neuroscience and Endocrinology, School of Clinical Sciences, University of Bristol, Whitson Street, Bristol BS1 3NY, UK. Electronic address: a.m.ocarroll@bristol.ac.uk.
authors: Anne-Marie
authors: AM
authors: O'Carroll
mesh_terms: D000075244:Apelin Receptors; D004705:Endocytosis / Q000187:drug effects*; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D013997:Time Factors
chemical_list: D000075244:Apelin Receptors; C491285:Aplnr protein, mouse; D036341:Intercellular Signaling Peptides and Proteins; D043562:Receptors, G-Protein-Coupled; C586143:apelin 13, Pyr(1)-; D048049:Extracellular Signal-Regulated MAP Kinases
keywords: Apelin; Apelin receptor; Extracellular-signal-regulated kinase (ERK); G protein-coupled receptor; Intracellular trafficking; Signalling