Na+/H+ Exchanger Regulatory Factor-1 Is Involved in Chemokine Receptor Homodimer CCR5 Internalization and Signal Transduction but Does Not Affect CXCR4 Homodimer or CXCR4-CCR5 Heterodimer

Abstract

Chemokine receptors are members of the G protein-coupled receptor (GPCR) family. CCR5 is also the principal co-receptor for macrophage-tropic strains of human immunodeficiency virus, type 1 (HIV-1), and efforts have been made to develop ligands to inhibit HIV-1 infection by promoting CCR5 receptor endocytosis. Given the nature of GPCRs and their propensity to form oligomers, one can consider ligand-based therapies as unselective in terms of the oligomeric composition of complexes. For example, a ligand targeting a CCR5 homomer could likely induce signal transduction on a heteromeric CCR5-CXCR4. Other avenues could therefore be explored. We identified a receptor adaptor interacting specifically with one receptor complex but not others. NHERF1, an adaptor known for its role in desensitization, internalization, and regulation of the ERK signaling cascade for several GPCRs, interacts via its PDZ2 domain with the CCR5 homodimer but not with the CXCR4-CCR5 heterodimer or CXCR4 homodimer. To further characterize this interaction, we also show that NHERF1 increases the CCR5 recruitment of arrestin2 following stimulation. NHERF1 is also involved in CCR5 internalization, as we demonstrate that coexpression of constructs bearing the PDZ2 domain can block CCR5 internalization. We also show that NHERF1 potentiates RANTES (regulated on activation normal T cell expressed and secreted)-induced ERK1/2 phosphorylation via CCR5 activation and that this activation requires NHERF1 but not arrestin2. Taken together, our results suggest that oligomeric receptor complexes can associate specifically with partners and that in this case NHERF1 could represent an interesting new target for the regulation of CCR5 internalization and potentially HIV infection.

Arrestin Scaffolds NHERF1 to the P2Y12 Receptor to Regulate Receptor Internalization

“…This study shows that P2Y 12 R internalization is NHERF1-dependent likely through an arrestin-dependent association with the extreme COOH terminus of this receptor. Whereas NHERF1 has previously been implicated in CCR5 internalization (33), this is the first report that NHERF1 expressed at endogenous levels is required for the internalization of a GPCR. The data presented here support recent reports of interactions between arrestin and NHERF1 (29,31,33) and furthermore, suggest that arrestin binding is required for NHERF1 recruitment to the activated receptor complex.…”

“…Whereas NHERF1 has previously been implicated in CCR5 internalization (33), this is the first report that NHERF1 expressed at endogenous levels is required for the internalization of a GPCR. The data presented here support recent reports of interactions between arrestin and NHERF1 (29,31,33) and furthermore, suggest that arrestin binding is required for NHERF1 recruitment to the activated receptor complex. In addition to providing insight into how the clinically relevant P2Y 12 R is regulated, these observations add another level of complexity to current models of NHERF-regulated GPCR function.…”

“…The internalization of the membrane multidrug resistance protein 4 (37) requires endogenous NHERF1. In addition, a recent study with the GPCR CCR5 showed that overexpressed NHERF1 increased the rate of CCR5 receptor internalization (33), suggesting that NHERF1 may play a wider role in receptor endocytosis. There appears to be key differences in the mechanism of NHERF1-regulated CCR5 regulation versus the P2Y 12 with the agonistindependent CCR5/NHERF1 interaction, whereas P2Y 12 -NH-ERF1 association was increased by agonist addition.…”

PDZ Protein Regulation of G Protein–Coupled Receptor Trafficking and Signaling Pathways

“…NHERF1 enhances chemokine (C-C motif) receptor 5 (CCR5) endocytosis and b-arrestin1 recruitment, thereby promoting the activation of ERK, Rho, and focal adhesion kinase signaling pathways, as well as potentially contributes to CCR5-mediated HIV-1 entry (Hammad et al, 2010;Kuang et al, 2012). NHERF1 overexpression also rescues the endocytosis of an internalization-defective platelet-activating factor receptor and antagonizes platelet-activating factor receptor-mediated inositol phosphate formation .…”

“…CCR5 functions as a coreceptor for HIV-1 viral entry into mammalian cells by functioning as a cofactor for the entry of the virus (Henrich and Kuritzkes, 2013). NHERF1 interactions with CCR5 function to enhance actin filament rearrangement of host cells, a function that is essential to allow postcell entry HIV-1 replication (Hammad et al, 2010;Kuang et al, 2012). PDZK1 interactions with hIPR selectively facilitate hIPRdependent activation of endothelial migration and vascular angiogenesis in vitro (Turner et al, 2011).…”