Agonist-induced internalization and desensitization of the apelin receptor. (5th December 2016)

• Record Type: Journal Article
• Authors: Pope, George R.; Tilve, Sharada; McArdle, Craig A.; Lolait, Stephen J.; O'Carroll, Anne-Marie
• Is Part Of: Molecular and cellular endocrinology. Volume 437(2016)
• Page Start: 108
• Page End: 119
• Publication Date: 2016-12-05
• Subjects: G protein-coupled receptor -- Apelin -- Apelin receptor -- Intracellular trafficking -- Signalling -- Extracellular-signal-regulated kinase (ERK); APJ apelin receptor -- βARR β-arrestin1 -- BIM bisindolylmaleimide I -- CME clathrin-mediated endocytosis -- CCV clathrin coated vesicle -- DNM dominant-negative mutant -- DYN dynamin -- eGFP enhanced green fluorescent protein -- FCS fetal calf serum -- GRK G protein-coupled receptor kinase 2 -- m mouse -- NGS normal goat serum -- P penicillin -- P70S6K S6 ribosomal protein kinase -- PCSE proportional cell surface expression -- PTX pertussis toxin -- S streptomycin -- U0126 1, 4-diamino-2, 3-dicyano-1, 4-bis[2-aminophenylthio] butadiene; Endocrinology -- Periodicals; Molecular biology -- Periodicals; Cytology -- Periodicals; Endocrinology -- Periodicals; Hormones -- Periodicals; Endocrinologie -- Périodiques; Cytology; Endocrinology; Molecular biology; Periodicals; 573.4
• DOI: 10.1016/j.mce.2016.07.040
• Languages: English
• ISSNs: 0303-7207

Abstract

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 [Pyr 1 ]apelin-13. The mechanisms of [Pyr 1 ]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 [Pyr 1 ]apelin-13 desensitized during sustained stimulation, due to upstream APJ-specific adaptive changes. Furthermore, [Pyr 1 ]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. Highlights: Agonist exposure induces internalization and reduction in cell surface APJ levels. ERK1/2 response to [Pyr 1 ]apelin-13 desensitizes rapidly during sustained stimulation. Desensitization of ERK1/2 response is due to upstream APJ-specific adaptive changes. Internalization is EPS15-, dynamin- and GRK2-dependent but β-arrestin-independent. Desensitization response is independent of dynamin, GRK2 and β-arrestin.