ERK1-Based Pathway as a New Selective Mechanism To Modulate CCR5 with Natural Antibodies

Abstract

Natural human Abs, recognizing an epitope within the first extramembrane loop of CCR5 (the main HIV coreceptor), induce a long-lasting internalization (48 h) of the protein, whereas all known CCR5 modulating molecules show a short-term kinetics (60–90 min). Despite extensive studies on the regulation of CCR5 signaling cascades, which are the effect of concomitant CCR5 internalization by exogenous stimuli such as Abs, downstream signaling continues to be poorly understood. In this article, we report a hitherto unrecognized mechanism of CCR5 modulation mediated by G protein–dependent ERK1 activity. We further demonstrate that ERK1 is localized mainly in the cytoplasmic compartment and that it interacts directly with the CCR5 protein, thus provoking possible CCR5 degradation with a subsequent de novo synthesis, and that re-expression of CCR5 on the cell membrane required several days. In contrast, the RANTES treatment induces a recovery of the receptor on the cell membrane in short-term kinetics without the involvement of de novo protein synthesis. The said new pathway could be relevant not only to better understand the molecular basis of all pathologic conditions in which CCR5 is involved but also to generate new tools to block viral infections, such as the use of recombinant Abs.

Class B β-arrestin2-dependent CCR5 signalosome retention with natural antibodies to CCR5

“…We analyzed a time course of anti-CCR5 Abs-induced signaling pathway after depleting cellular levels of β-arrestin1 or -2 or both by nucleofecting siRNAs specific to each isoform. The experiments were performed in the T lymphoblastoid R5-SupT1-L23 cell line, which has a CCR5 cell surface level and an activation status comparable to human CD4+ T lymphocytes2124. The activation status of the cells has been evaluated by flow cytometry and the mean fluorescence (MFI) obtained with anti-CD38 antibody was 54.5 and 34.5 respectively, as previously reported21.…”

“…Our group has recently demonstrated that CCR5 specific antibodies (CCR5 Ab Pos), found in Long Term HIV infected subjects (LTNPs), induce a long lasting internalization of the receptor (48 h) mediated by G-protein-dependent ERK1 activity, both in R5-SupT1-L23 and in CD4+ T lymphocytes21. Instead, other groups have demonstrated that when CCR5 internalization is mediated by RANTES, the level of receptor on the cell surface recovered to nearly 100% after 120′ incubation42223.…”

“…Instead, other groups have demonstrated that when CCR5 internalization is mediated by RANTES, the level of receptor on the cell surface recovered to nearly 100% after 120′ incubation42223. Taking into account that anti-CCR5 Abs lead to a statistically significant accumulation of β-arrestin1/2 as well21, we performed an immunoprecipitation assay to evaluate the interaction among the components and the temporal distribution of the CCR5 signalosome. R5-SupT1-M10 cell line, which displays medium levels of CCR5 expression24, was used to immunoprecipitate both CCR5 and β-arrestin1/2.…”

“…R5-SupT1-M10 cell line, which displays medium levels of CCR5 expression24, was used to immunoprecipitate both CCR5 and β-arrestin1/2. We used a pool of 5 LTNPs sera, each containing CCR5 antibodies, or not (named CCR5 Ab Neg and used as a negative control), as previously described and characterized by our group2125; we also used the chemokine RANTES, as a positive control26. In detail, the cells were incubated for 30′ with Rantes and with CCR5 Ab Pos or not, washed and incubated for a further 120′ (i.e., incubation for this group totaled 150′), t0, and for 48 h (t1); these two time points have been chosen on the basis of our previous findings21; briefly, 150′ corresponds to the initial internalization of the receptor and 48 h is required to achieve complete CCR5 internalization.…”

“…Recently, we reported an hitherto unrecognized mechanism of CCR5 modulation mediated by G-protein-dependent ERK1 activity upon stimulation with anti CCR5 natural antibodies21, that promotes cytoplasmatic retention of phosphorylated ERK1.…”