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.

Free fatty acid receptor 1 agonist, MR1704, lowers blood glucose levels in rats unresponsive to the sulfonylurea, glibenclamide

“…It is thus reasonable to assume that the lack of releasable insulin, owing to the reduced β cell insulin content exacerbates glucose tolerance. Although many GPCRs are rapidly desensitized after agonist binding (Min, Zheng, Zhang, Caron, & Kim, ; Mohammad et al, ; Pope, Tilve, McArdle, Lolait, & O'carroll, ; Robinett, Deshpande, Malone, & Liggett, ), GPR40/FFAR1 activation by MR1704 did not cause desensitization, which was similar to that found for receptor activation by fasiglifam in diabetic neonatally streptozotocin‐induced rats (Ito et al, ). The sustained glucose‐lowering effects of MR1704 in the present model support the idea that this type of GPR40/FFAR1 agonist have a low risk for tachyphylaxis or pancreatic β cell exhaustion compared long‐term treatment with SUs.…”

Expression and functional implications of the renal apelinergic system in rodents

“…Dose-response curves were performed for 10min, where [Pyr 1 ]apelin-13 stimulation of ERK1/2 is maximal as established in pilot studies on CHO-B78 cells. After incubation with 0.01-1000nM [Pyr 1 ]apelin-13 (Bachem) or rat apela-32 (cyclized, pyroglutamated; Severn Biotech, U.K.), or vehicle (dH 2 O), the cells were immunostained for dual phosphorylated(pp) ERK1/2 and total(t) ERK1/2—cell images were acquired using the IN Cell Analyzer 1000 and analysis performed by In Cell Analyzer Workstation 3.5 software (GE Healthcare) as previously described [ 27 , 28 ]. The nuclear and cytoplasmic fluorescence intensities in individual cells were quantified (in arbitrary units)—data was normalized to vehicle controls after subtraction of ‘no primary antibody’ backgrounds.…”

International Union of Basic and Clinical Pharmacology. CVII. Structure and Pharmacology of the Apelin Receptor with a Recommendation that Elabela/Toddler Is a Second Endogenous Peptide Ligand

“…A further limitation of endogenous apelin such as [Pyr 1 ]apelin-13 is that, in addition to activating G proteins, signaling involves coupling to β -arrestin (Evans et al, 2001), resulting in receptor desensitization (Masri et al, 2006, and has be characterized in detail by Pope et al (2016). This reduces the efficacy of repeated dosing by apelin ligands.…”