Different Mechanisms Regulate Lysophosphatidic Acid (LPA)-dependent Versus Phorbol Ester-dependent Internalization of the LPA1 Receptor

Nikhil M Urs,Harish Radhakrishna,Andrew P Kowalczyk

doi:10.1074/jbc.m710003200

Abstract

Lysophosphatidic acid (LPA) stimulates cells by activation of five G-protein-coupled receptors, termed LPA 1-5. The LPA 1 receptor is the most widely expressed and is a major regulator of cell migration. In this study, we show that phorbol ester (PMA)-induced internalization of the LPA(1) receptor requires clathrin AP-2 complexes, protein kinase C, and a distal dileucine motif (amino acids 352 and 353) in the cytoplasmic tail but not beta-arrestin. Agonist-dependent internalization of LPA 1, however, requires a cluster of serine residues (amino acids 341-347) located proximal to the dileucine motif, beta-arrestin, and to a lesser extent clathrin AP-2. The serine cluster of LPA 1 is required for beta-arrestin2-GFP translocation to the plasma membrane and signal desensitization. In contrast, the dileucine motif (IL) is required for both basal and PMA-induced internalization. Evidence for the beta-arrestin independence of PMA-induced internalization of LPA 1 comes from the observations that beta-arrestin2-GFP is not recruited to the plasma membrane upon PMA treatment and that LPA 1 is readily internalized in beta-arrestin1/2 knock-out mouse embryonic fibroblasts. These results indicate that distinct molecular mechanisms regulate agonist-dependent and PMA-dependent internalization of the LPA 1 receptor.

Highlights

Adaptor proteins that are involved in the clathrin-mediated internalization of GPCRs include clathrin, AP-2, and ␤-arrestins [2, 8, 9]. ␤-Arrestins bind to many GPCRs (e.g. ␤2AR, LPA1, etc.) [2, 7] at specific serine/threonine residues, which have been preferentially phosphorylated by G-protein receptor kinases [2]
In addition to lysophosphatidic acid (LPA), a previous study indicated that the phorbol ester, Phorbol 12-myristate 13-acetate (PMA), induced PKC-dependent phosphorylation, signal desensitization, and internalization of the LPA1 Receptor (LPA1R) in C9 rat hepatoma cells [27]
We have previously showed that LPA1Rs transiently associate with ␤-arrestins at the plasma membrane and that this association is required for signal attenuation [7]

Summary

Introduction

Adaptor proteins that are involved in the clathrin-mediated internalization of GPCRs include clathrin, AP-2, and ␤-arrestins [2, 8, 9]. ␤-Arrestins bind to many GPCRs (e.g. ␤2AR, LPA1, etc.) [2, 7] at specific serine/threonine residues, which have been preferentially phosphorylated by G-protein receptor kinases [2]. We have previously shown that the LPA1 receptor utilizes a clathrin- and ␤-arrestin-dependent pathway for agonist-induced internalization [7, 19]. Two distinct motifs are required for agonistdependent versus agonist-independent internalization of the LPA1 receptor. A serine cluster (341SDRSASS347) in the C-terminal tail is required for ␤-arrestin association, signal attenuation, and subsequent endocytosis after LPA stimulation. A more distal dileucine motif (352IL353) is required for agonistindependent and phorbol ester-induced internalization of the LPA1 receptor. This type of internalization is independent of ␤-arrestin but requires the clathrin adaptor AP-2. This suggests that two distinct mechanisms regulate agonist-dependent and PMA-dependent internalization of the LPA1 receptor

Methods

Results

Conclusion