Abstract
Phosphorylation of G-protein-coupled receptors (GPCRs) by GRKs and subsequent recruitment of beta-arrestins to agonist-occupied receptors serves to terminate or attenuate signaling by blocking G-proteins from further interaction with the receptors. Human cytomegalovirus encodes a GPCR termed US28 that is homologous to the human chemokine family of GPCRs but differs from the cellular receptors in that it maintains high constitutive activity in the absence of agonist. Although US28 is constitutively active, mechanisms that regulate this activity are unknown. We provide evidence that US28 is constitutively phosphorylated by GRKs in cells and that in consequence, beta-arrestin 2 is localized to the plasma membrane. Deletion of the carboxyl terminal 40 amino acids in US28 generates a receptor that is severely impaired in its ability to become phosphorylated and recruit beta-arrestin and accordingly demonstrates increased inositol phosphate signaling. This result indicates that the carboxyl terminus of US28 contains an important signaling regulatory region and mutational analysis deleting carboxyl terminal serines identified serine 323 as a critical residue within this region. In addition, overexpression of wild type GRK5 leads to hyperphosphorylation of US28 that results in a decrease of inositol phosphate accumulation. These results are consistent with the hypothesis that GRK phosphorylation and recruitment of beta-arrestin to the US28 viral GPCR attenuates signaling to the traditional Galphaq-stimulated inositol phosphate pathway. Finally, in contrast to the results with inositol phosphate signaling, we provide evidence that the US28 carboxyl-terminal phosphorylation sites and beta-arrestin-interacting domain are required for maximal activation of the p38 mitogen-activated protein kinase. Taken together, these results indicate that US28 interacts with these important regulatory proteins to control multiple aspects of signal transmission. Understanding the regulation of viral GPCRs by GRKs and beta-arrestins will provide important new insights into not only aspects of viral pathogenesis but also basic mechanisms of receptor signaling.
Highlights
G-protein-coupled receptors (GPCRs)1 constitute a family of seven-transmembrane domain proteins that transmit signals to the interior of the cell by activating a variety of signaling pathways [1,2,3]
human cytomegalovirus (HCMV) US28 Is Constitutively Phosphorylated in the Absence of Agonist—Stimulation of cellular GPCRs with agonist leads to a rapid and transient increase in signaling activity
The HCMV-encoded US28 GPCR has been shown to maintain strong constitutive signaling capacity in the absence of agonist, it remains unknown whether phosphorylation and -arrestin binding regulate this signaling
Summary
Cell Culture and Transfection—All cell lines were obtained from the American Type Culture Collection (ATCC). HEK-293 cells were transfected in 100-mm dishes with 5 g of total plasmid DNA using a modified calcium phosphate method. [32P]Orthophosphate Labeling and Receptor Immunoprecipitations—48 h post-transfection, cells were starved in phosphate-free Dulbecco’s modified Eagle’s medium supplemented with 2% dialyzed fetal bovine serum and 20 mM HEPES for 30 min. 48 h after transfection, medium was aspirated, and cells were incubated with anti-FLAG primary antibody diluted 1:500 in PBS on ice for 2 h, washed once in PBS, and incubated with fluorescein isothiocyanate-conjugated antimouse antibody diluted 1:250 in PBS on ice for 1 h. Cells were fixed in phosphate-buffered saline containing 4% formaldehyde, and the presence of cell-surface receptors was determined by flow cytometry (Duke University Flow Cytometry Facility). For confocal microscopic localization of -arrestin, HEK-293 cells were transfected with the appropriate combination of -arrestin 2-GFP, US28, and GRK plasmids. For confocal microscopic localization of -arrestin, HEK-293 cells were transfected with the appropriate combination of -arrestin 2-GFP, US28, and GRK plasmids. 24 h after transfection, cells were transferred into collagen-coated glass bottom dishes. 48 h after transfection confocal microscopy was performed on a Zeiss LSM510 laser-scanning microscope using a ϫ 100/1.4 numerical aperture oil immersion lens
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