β-arrestin1 and distinct CXCR4 structures are required for stromal derived factor-1 to downregulate CXCR4 cell-surface levels in neuroblastoma

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 β-arrestin1, we showed that β-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 β-arrestin1 expression and the 343-352 region. Together, these results characterize CXCR4 structural domains and β-arrestin1 as critical regulators of CXCR4 cell-surface expression in neuroblastoma. β-Arrestin1 levels may therefore influence the CXCR4-driven metastasis of neuroblastoma as well as prognosis.