Aquaporin-4 (AQP4) water channel-specific IgG distinguishes neuromyelitis optica (NMO) from multiple sclerosis and causes characteristic immunopathology in which central nervous system (CNS) demyelination is secondary. Early events initiating the pathophysiological outcomes of IgG binding to astrocytic AQP4 are poorly understood. CNS lesions reflect events documented in vitro following IgG interaction with AQP4: AQP4 internalization, attenuated glutamate uptake, intramyelinic edema, interleukin-6 release, complement activation, inflammatory cell recruitment, and demyelination. Here, we demonstrate that AQP4 internalization requires AQP4-bound IgG to engage an astrocytic Fcγ receptor (FcγR). IgG-lacking Fc redistributes AQP4 within the plasma membrane and induces interleukin-6 release. However, AQP4 endocytosis requires an activating FcγR's gamma subunit and involves astrocytic membrane loss of an inhibitory FcγR, CD32B. Interaction of the IgG-AQP4 complex with FcγRs triggers coendocytosis of the excitatory amino acid transporter 2 (EAAT2). Requirement of FcγR engagement for internalization of two astrocytic membrane proteins critical to CNS homeostasis identifies a complement-independent, upstream target for potential early therapeutic intervention in NMO.N euromyelitis optica (NMO) is a relapsing inflammatory autoimmune demyelinating disease of the central nervous system (CNS), long considered a severe form of multiple sclerosis (MS). Attacks are generally more severe, onset of paralysis and blindness more rapid (1), and standard MS therapies can worsen NMO (2, 3). A specific serum IgG allows early distinction from MS, thus ensuring timely appropriate therapy (4, 5).The aquaporin-4 (AQP4) water channel is the CNS target (4). Outcomes documented in vitro after NMO-IgG binds to the AQP4 extracellular domain on astrocytes include AQP4 endocytosis and lysosomal degradation, loss of its physically linked major excitatory amino acid transporter 2 (EAAT2), reduced glutamate uptake, impaired water fluxes, granulocyte chemotaxis, and complement factor transcription, secretion, and activation (1, 6-11). Ensuing endothelial permeation of circulating immunoglobulins, complement components, and leukocytes magnifies the initial inflammatory response (6-12). Immunopathological analyses of NMO patients' CNS tissues attest to these events occurring in vivo and inform lesional evolution at the target astrocyte level (1). The blood-brain barrier (BBB) does not absolutely restrict IgG entry into the CNS, but it does exclude macromolecular C1q, essential for activating the classical complement cascade.Experimental studies of lesional events in vitro and in animal models of NMO have emphasized complement-mediated inflammation and astrocyte cytolysis, late events associated with extensive BBB disruption. A neglected potential initiator of NMO pathophysiology is the AQP4-IgG-activated astrocyte's response: synthesis and secretion of complement, cytokines, chemokines (12-14), and inflammatory mediators attracting NMO-characteristic gra...

The CXCR4 chemokine receptor is a G protein-coupled receptor (GPCR) that signals in T lymphocytes by forming a heterodimer with the T cell antigen receptor (TCR). CXCR4 and TCR functions are consequently highly cross-regulated, affecting T cell immune activation, cytokine secretion, and T cell migration. The CXCR4-TCR heterodimer stimulates T cell migration and activation of the ERK MAP kinase and downstream AP-1-dependent cytokine transcription in response to SDF-1, the sole chemokine ligand of CXCR4. These responses require Gi-type G proteins as well as TCR ITAM domains and the ZAP-70 tyrosine kinase, thus indicating that the CXCR4-TCR heterodimer signals to integrate GPCR-associated and TCR-associated signaling molecules in response to SDF-1. Yet, the phospholipase C (PLC) isozymes responsible for coupling the CXCR4-TCR heterodimer to distinct downstream cellular responses are incompletely characterized. Here, we demonstrate that PLC activity is required for SDF-1 to induce ERK activation, migration, and CXCR4 endocytosis in human T cells. SDF-1 signaling via the CXCR4-TCR heterodimer uses PLC-β3 to activate the Ras-ERK pathway and increase intracellular Ca2+ concentrations, while PLC-γ1 is dispensable for these outcomes. In contrast, PLC-γ1, but not PLC-β3, is required for SDF-1-mediated migration, via a mechanism independent of LAT. These results increase understanding of the signaling mechanisms employed by the CXCR4-TCR heterodimer, characterize new roles for PLC-β3 and PLC-γ1 in T cells, and suggest that multiple PLCs may also be activated downstream of other chemokine receptors in order to distinctly regulate migration versus other signaling functions.

CXC chemokine receptor 4 (CXCR4) is a G protein-coupled receptor (GPCR) located on the cell surface that signals upon binding the chemokine stromal derived factor-1 (SDF-1; also called CXCL 12). CXCR4 promotes neuroblastoma proliferation and chemotaxis. CXCR4 expression negatively correlates with prognosis and drives neuroblastoma growth and metastasis in mouse models. All functions of CXCR4 require its expression on the cell surface, yet the molecular mechanisms that regulate CXCR4 cell-surface levels in neuroblastoma are poorly understood. We characterized CXCR4 cell-surface regulation in the related SH-SY5Y and SK-N-SH human neuroblastoma cell lines. SDF-1 treatment caused rapid down-modulation of CXCR4 in SH-SY5Y cells. Pharmacologic activation of protein kinase C similarly reduced CXCR4, but via a distinct mechanism. Analysis of CXCR4 mutants delineated two CXCR4 regions required for SDF-1 treatment to decrease cell-surface CXCR4 in neuroblastoma cells: the isoleucine-leucine motif at residues 328 and 329 and residues 343-352. In contrast, and unlike CXCR4 regulation in other cell types, serines 324, 325, 338, and 339 were not required. Arrestin proteins can bind and regulate GPCR cell-surface expression, often functioning together with kinases such as G protein-coupled receptor kinase 2 (GRK2). Using SK-N-SH cells which are naturally deficient in b-arrestin1, we showed that b-arrestin1 is required for the CXCR4 343-352 region to modulate CXCR4 cell-surface expression following treatment with SDF-1. Moreover, GRK2 overexpression enhanced CXCR4 internalization, via a mechanism requiring both b-arrestin1 expression and the 343-352 region. Together, these results characterize CXCR4 structural domains and b-arrestin1 as critical regulators of CXCR4 cell-surface expression in neuroblastoma. b-Arrestin1 levels may therefore influence the CXCR4-driven metastasis of neuroblastoma as well as prognosis.

IQGAP1 mediates CXCR4 cell surface expression and signaling by regulating EEA-1+ endosome interactions with microtubules during CXCR4 trafficking and recycling.