Human cytomegalovirus-encoded G protein-coupled receptor US28 mediates smooth muscle cell migration through Galpha12

Abstract

Coupling of G proteins to ligand-engaged chemokine receptors is the paramount event in G-protein-coupled receptor signal transduction. Previously, we have demonstrated that the human cytomegalovirus-encoded chemokine receptor US28 mediates human vascular smooth muscle cell (SMC) migration in response to either RANTES or monocyte chemoattractant protein 1. In this report, we identify the G proteins that couple with US28 to promote vascular SMC migration and identify other signaling molecules that play critical roles in this process. US28-mediated cellular migration was enhanced with the expression of the G-protein subunits Galpha12 and Galpha13, suggesting that US28 may functionally couple to these G proteins. In correlation with this observation, US28 was able to activate RhoA, a downstream effector of Galpha12 and Galpha13 in cell types with these G proteins but not in those without them and activation of RhoA was dependent on US28 stimulation with RANTES. In addition, inactivation of RhoA or the RhoA-associated kinase p160ROCK with a dominant-negative mutant of RhoA or the small molecule inhibitor Y27632, respectively, abrogated US28-induced SMC migration. The data presented here suggest that US28 functionally signals through Galpha12 family G proteins and RhoA in a ligand-dependent manner and these signaling molecules are important for the ability of US28 to induce cellular migration.

FIG. 1.

Gα-protein detection in human vascular SMCs and rat AoSMCs. Shown are representative Western blots of Gα-protein expression in human PASMCs and rat AoSMCs and a table illustrating the presence of Gα subunits expressed in various HCMV-permissive cell types, including PASMCs, CASMCs, U373MG, and NHDFs as well as cell types commonly used in signal transduction studies, COS7 and rat AoSMCs.

FIG. 2.

Gα12/13 enhances US28-mediated SMC migration. PASMCs were infected with HCMV and adenoviruses (Ad) expressing Gαq, Gα11, Gα12, Gα13, or Gαi1. Expression of the Gα proteins was normalized by Western blotting, and the MOI was adjusted accordingly such that equivalent amounts of protein were expressed. The total number of cells migrating from the upper to lower chamber was enumerated at 48 to 72 h postinfection.

FIG. 3.

US28 activates RhoA in a ligand-dependent manner. (A) The ability of US28 to induce migration of rat AoSMCs was determined by infecting cells with adenoviruses (Ad) expressing US28 in the presence of 10-ng/ml human, rat, or mouse RANTES. The number of cells migrating was determined by microscopic enumeration at 48 to 72 h postinfection. (B) Rat AoSMCs were serum starved for 16 to 18 h and subsequently infected with adenoviruses expressing US28. US28-expressing cells were either unstimulated or stimulated with 1- or 50-ng/ml RANTES at 16 to 18 h postinfection. At the indicated times, cells were harvested and lysates were applied to GST-Rhotekin-baited beads in order to precipitate active RhoA. The amount of bound/active RhoA was determined by Western blotting with an anti-RhoA polyclonal antibody. To ensure equal loading, lysates were also probed for total RhoA prior to being applied to GST-Rhotekin beads (lower panel). (C) US28-induced RhoA activity assay performed in U373MG cells as described above for rat AoSMCs.

FIG. 4.

Reconstitution of NHDFs with Gα12 restores US28-induced RhoA activation. (A) COS7 cells were serum starved for 18 h and then infected with adenoviruses expressing US28 and/or Ad-trans. Cells were stimulated at 18 h postinfection with 25-ng/ml RANTES for 0 (unstimulated), 5, 10, or 30 min. Alternatively, serum-starved COS7 cells were stimulated with 10-ng/ml platelet-derived growth factor (PDGF)-BB or 1% serum for 10 or 30 min. Active RhoA assays were performed, and RhoA was visualized by SDS-PAGE and Western blotting. The amount of total input RhoA was also assessed by Western blotting (lower lanes). (B) NHDFs were serum starved and infected as described above. Infected cells were stimulated with 1- to 50-ng/ml RANTES for 0, 5, 10, or 30 min or with 1% serum for 10 min (positive control), and RhoA activity was determined as described above. (C) NHDFs were infected with adenoviruses expressing either Gα12 and/or US28 and then treated with 10-ng/ml RANTES for 10 min.

FIG. 5.

RhoA activity is essential for US28-induced SMC migration. PASMCs were infected with HCMV along with adenoviruses (Ad) expressing either WT or DN RhoA at the indicated MOI. Cellular migration was determined by microscopy at 48 to 72 h postinfection. Cellular migration is expressed as a percentage when compared to the number of cells migrating when coinfected with HCMV and Ad-Trans.

FIG. 6.

p160ROCK activity is important for US28-mediated actin cytoskeleton reorganization and cellular migration. (A) PASMCs expressing US28 were left untreated or were treated with the p160ROCK inhibitor Y27632. RANTES-treated cells were fixed 4 h post-ligand addition. Cells were stained for actin with phalloidin (red), the US28 flag epitope, and for nuclei with Hoechst DNA staining dye. (B) PASMCs were infected with HCMV and then treated with increasing concentrations of the p160ROCK inhibitor Y27632. The number of cells migrating from the upper to lower chamber was enumerated by microscopy and is expressed as a total number of cells migrating from the upper to lower chamber.

FIG. 7.

US28 signaling through Gα12 family G proteins. Upon ligand binding, US28 couples with Gα12/13 in vascular SMCs. Gα12 family G proteins stimulate RhoGEFs, which activate RhoA through promoting the exchange of GDP for GTP. Active RhoA plays a critical role in the actin cytoskeleton rearrangements that are necessary for US28-induced SMC migration. RANTES stimulation of US28 also leads to the activation of the RhoA effector p160ROCK, which is essential in US28-mediated SMC migration.