Human Cytomegalovirus-Encoded G Protein-Coupled Receptor US28 Mediates Smooth Muscle Cell Migration through Gα12

Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus with an incidence of infection ranging between 40 and 100%, depending on age and socioeconomic status of the infected individual. Infection generally occurs during childhood, and after primary infection, HCMV establishes a lifelong persistence in the host. HCMV infection of healthy immunocompetent individuals is commonly asymptomatic. However, HCMV may cause life-threatening disease in immunocompromised patients (6), and HCMV is the leading cause of birth defects in virally infected neonates (11). HCMV has also been linked to long-term diseases, including atherosclerosis, arterial restenosis following angioplasty, and transplant vascular sclerosis associated with chronic allograft rejection (22-24, 26, 30, 31). A hallmark of all of these diseases is the migration of arterial smooth muscle cells (SMCs) from the vessel media to the intima, ultimately resulting in vessel occlusion. HCMV infection of SMCs in vivo has been linked to a viral etiology of vascular disease. Previously, we have demonstrated that HCMV infection of primary arterial but not venous SMCs results in significant cellular migration (32). Similar to other herpesviruses, HCMV encodes chemokine receptor homologues, including UL33, UL78, US27, and US28. Ablation of one of these HCMV-encoded chemokine receptors, US28, within the virus, abrogates SMC migration, which is rescued only by expression of the viral homologue and not the cellular chemokine receptor CCR5 (32). US28 is structurally similar to the human chemokine receptor CCR1 (12) and binds chemokines of the β-class (CC), including monocyte chemoattractant protein-1 (MCP-1), MCP-3, RANTES, macrophage inflammatory polypeptides 1α (MIP-1α) and MIP-1β (5, 19, 29), and the CX3C chemokine fractalkine (15, 18). Expression of US28 in the presence of CC chemokines, including RANTES or MCP-1, was sufficient to promote SMC migration, which was inhibited by protein tyrosine kinase (PTK) inhibitors but not pertussis toxin (32). Recently, our group has demonstrated that US28 signals through the nonreceptor PTKs Src and focal adhesion kinase (FAK) and that this activity is necessary for US28-mediated SMC migration (33). US28-mediated SMC migration was inhibited by treatment with the Src inhibitor PP2, and through the expression of either of two dominant-negative (DN) inhibitors of FAK. US28 is the first viral G-protein-coupled receptor (GPCR) to be shown to mediate cellular movement, which is cell type specific and provides a molecular basis for the correlative evidence that links HCMV to the acceleration of vascular disease. The first link between GPCRs and the intracellular signaling milieu are the heterotrimeric G proteins, consisting of a complex of α, β, and γ subunits. The Gα proteins are subdivided into one of four different subfamilies based on sequence similarities: Gαi, Gαq, Gα12, or Gαs. Upon ligand binding, GPCRs undergo conformational changes that promote interactions with α subunits of the heterotrimeric G-protein complex, which causes the exchange of GDP for GTP, initiating intracellular signaling cascades. The Gαi family (Gαi1, Gαi2, Gαi3, Gαo, and Gαz) has a wide tissue distribution and is pertussis toxin (PTX) sensitive. The only exceptions are Gαo and Gαz, which are PTX insensitive and limited to neuronal tissues (both Gαo and Gαz) and platelets (Gαz only) (10). Numerous hormones and inflammatory cytokines signal through Gαi pathways, as is evidenced by their PTX sensitivity (10, 25, 34). Signaling pathways activated by the ubiquitously expressed Gαs often stimulate the accumulation of cyclic AMP (cAMP) (10). Gαq family members activate a diverse array of signaling pathways that includes the activation of phospholipase C-β (PLC-β), producing the second messenger molecules inositol (1,4,5)-triphosphate and diacylglycerol. Gαq and Gα11 are ubiquitously expressed, while the other family members Gα14 (kidney, lung, spleen, and testis) and Gα15/16 (myeloid and lymphoid lineage cells) display a more limited tissue distribution (10). Gα12 family members Gα12 and Gα13 exhibit a ubiquitous tissue distribution (10) and interact with a number of different RhoGEFs stimulating robust RhoA signaling activity (7, 13, 14). Cellular migration is an important process in a number of physiological responses, including leukocyte trafficking, the formation of secondary lymphoid organs, and remodeling of the vasculature. The most-well-characterized migratory cell type is leukocytes migrating in response to chemokines. The process of leukocyte migration and homing to secondary lymphoid tissues is highly complex, involving a multitude of chemokine receptors and chemokines from most of the chemokine families. In general, leukocyte migration induced by chemokine receptors is sensitive to PTX, suggesting a role for Gαi family G proteins in this process (2, 34). US28 has been shown to couple to a variety of different G proteins, including Gαi/o, Gα16, and Gαq/11, and different aspects of US28 signaling have been demonstrated to be either PTX sensitive or insensitive (4, 8). However, US28-mediated SMC migration has been shown to be insensitive to PTX, suggesting that Gαi is not involved in this event (32). Therefore, we examined the signaling components involved in US28-induced SMC migration. In this paper, we demonstrate that US28-induced SMC migration involves signaling through the Gα12 pathway.