The fate of the internalized apelin receptor is determined by different isoforms of apelin mediating differential interaction with β-arrestin

doi:10.1016/j.bbrc.2010.03.151

Biochemical and Biophysical Research Communications

Volume 395, Issue 2, 30 April 2010, Pages 185-189

https://doi.org/10.1016/j.bbrc.2010.03.151 Get rights and content

Abstract

Internalization of the apelin receptor by apelin-13 is characterized by dissociation from β-arrestins and rapid recycling to the cell surface. Paradoxically, the apelin receptor internalized by apelin-36 was sequestered intracellularly. The specific pathways involved in apelin receptor trafficking were resolved using β-arrestin1 and constitutively active and dominant negative Rab proteins following activation by apelin-13 or apelin-36. β-Arrestin1 dissociated from the apelin-13-internalized receptor while the apelin-36-internalized receptor was trafficked with β-arrestin1 to intracellular compartments. The apelin-13-internalized receptor was rapidly recycled to the cell surface through a Rab4-dependent mechanism while Rab7 targeted the receptor to lysosomes. The internalized receptor co-expressed with dominant negative Rab4 were trafficked to lysosomes. These observations revealed a novel ligand-dependent targeting of the apelin receptor to β-arrestin-associated and -dissociated trafficking pathways and a role for different Rab proteins to direct these pathways.

Introduction

The receptor for apelin is a member of the G protein-coupled receptor (GPCR) family, the largest class of cell surface receptor proteins. Apelin is a potent cardiac inotropic agent, lowers blood pressure, modulates pituitary hormone release, stimulates water intake, is involved in stress activation and has been identified as a novel adipokine secreted from fat cells to regulate insulin (reviewed in [1], [2]) The apelinergic system has garnered much attention as a target for novel drug discovery [3] for pathophysiological conditions involving these systems. Apelin was first purified as a 36 amino acid peptide and can be cleaved to a smaller 13 amino acid bioactive peptide [4]. Apelin-13 and apelin-36 differ in tissue distribution [5], receptor binding affinity, and their ability to effect the intracellular trafficking of the apelin receptor [4], [6]. Apelin-13 has greater affinity and potency for the apelin receptor than apelin-36, although the dissociation rate of apelin-36 from the apelin receptor was observed to be dramatically lower than apelin-13. This distinction in the rate of dissociation may account for observed differences in the duration of apelin-13 and apelin-36-induced responses.

The internalization of GPCRs is an important process for the regulation of GPCR signaling. GPCR internalization is primarily mediated by a clathrin-mediated mechanism, usually following receptor phosphorylation by GPCR kinases (GRKs) [7]. GRK phosphorylation promotes recruitment of β-arrestins to GPCRs and targets them for internalization. Apelin-13-activated receptors recruit both β-arrestin1 and β-arrestin2 to the cell surface, do not internalize as a complex with β-arrestins and recycle rapidly back to the cell surface suggesting that the apelin receptor is a “Class A” GPCR [8], [9]. In contrast, apelin-36 treatment results in the intracellular sequestration of apelin receptors even after 2 h of apelin-36 washout [10]. These findings suggest ligand-dependent differential trafficking pathways for the apelin receptor. The mechanisms underlying these divergent pathways of apelin receptor trafficking have not yet been elucidated.

In this study, we delineate the endocytotic pathways involved in the trafficking of the apelin receptor following activation by apelin-13 or apelin-36 and have defined the roles for both β-arrestin1 and Rab GTPase proteins in this process. We show that, rather than being the property of the receptor, it is the identity of the endogenous agonist that dictated the association of the apelin receptor with β-arrestin1, and that depending on this association the receptor was routed into two divergent trafficking pathways.

Section snippets

Construction of cDNA

DNA encoding the green fluorescent protein (GFP) fused proteins GFP-Rab4, GFP-Rab4(N121I), GFP-Rab5, GFP-Rab7, GFP-Rab7(N125I), GFP-Rab7(Q67L), GFP-Rab11, GFP-Rab11(Q70L) and β-arrestin1-GFP constructs were obtained as previously described [11]. The cDNA encoding monomeric red fluorescent protein (mRFP) was a gift from Dr. Roger Tsien (University of California, San Diego, CA). The stop codon of cDNA encoding the human apelin receptor was modified to contain BamHI or KpnI sites by PCR

Differential trafficking of the apelin receptor following activation by apelin-13 or apelin-36

To investigate the intracellular trafficking of internalized apelin receptor following agonist treatment, we created HEK293 cell lines that stably expressed the apelin receptor fused to green fluorescent protein (GFP) or to monomeric red fluorescent protein (mRFP). In the absence of agonist apelin receptor-GFP was localized at the cell surface, with some perinuclear expression detected (Fig. 1A). In response to agonist treatment with 500 nM of either apelin-13 or apelin-36 for 1 h, the apelin

Discussion

In this study, we investigated the mechanisms delineating the differential trafficking of the apelin receptor by its endogenous ligands apelin-13 and apelin-36 and the role of β-arrestin1 and Rab GTPases to direct apelin receptor trafficking. Our findings revealed that the apelin-13-internalized receptor dissociated from β-arrestin1 prior to receptor endocytosis, while the apelin-36-internalized receptor remained associated with β-arrestin1 and trafficked together to early endosomes. Using

Acknowledgments

This research was funded by a Canadian Institutes of Health Research (CIHR) grant to S.R.G. and B.F.O. S.R.G. is the recipient of a Canada Research Chair.

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Citation Excerpt :
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The fate of the internalized apelin receptor is determined by different isoforms of apelin mediating differential interaction with β-arrestin

Abstract

Introduction

Section snippets

Construction of cDNA

Differential trafficking of the apelin receptor following activation by apelin-13 or apelin-36

Discussion

Acknowledgments

Trends Pharmacol. Sci.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Life Sci.

J. Biol. Chem.

Virology

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Life Sci.

Biochim. Biophys. Acta