The human cytomegalovirus chemokine receptor US28 mediates vascular smooth muscle cell migration

Human cytomegalovirus (HCMV) infection of smooth muscle cells (SMCs) in vivo has been linked to a viral etiology of vascular disease. In this report, we demonstrate that HCMV infection of primary arterial SMCs results in significant cellular migration. Ablation of the chemokine receptor, US28, abrogates SMC migration, which is rescued only by expression of the viral homolog and not a cellular G protein-coupled receptor (GPCR). Expression of US28 in the presence of CC chemokines including RANTES or MCP-1 was sufficient to promote SMC migration by both chemokinesis and chemotaxis, which was inhibited by protein tyrosine kinase inhibitors. US28-mediated SMC migration provides a molecular basis for the correlative evidence that links HCMV to the acceleration of vascular disease.     

Human cytomegalovirus chemokine receptor US28-induced smooth muscle cell migration is mediated by focal adhesion kinase and Src

The human cytomegalovirus-encoded chemokine receptor US28 induces arterial smooth muscle cell (SMC) migration; however, the underlying mechanisms involved in this process are unclear. We have previously shown that US28-mediated SMC migration occurs by a ligand-dependent process that is sensitive to protein-tyrosine kinase inhibitors. We demonstrate here that US28 signals through the non-receptor protein-tyrosine kinases Src and focal adhesion kinase (FAK) and that this activity is necessary for US28-mediated SMC migration. In the presence of RANTES (regulated on activation normal T cell expressed and secreted), US28 stimulates the production of a FAK.Src kinase complex. Interestingly, Src co-immunoprecipitates with US28 in a ligand-dependent manner. This association occurs earlier than the formation of the FAK.Src kinase complex, suggesting that US28 activates Src before FAK. US28 binding to RANTES also promotes the formation of a Grb2.FAK complex, which is sensitive to treatment with the Src inhibitor PP2, further highlighting the critical role of Src in US28 activation of FAK. Human cytomegalovirus US28-mediated SMC migration is inhibited by treatment with PP2 and through the expression of either of two dominant negative inhibitors of FAK (F397Y and NH2-terminal amino acids 1-401). These findings demonstrate that activation of FAK and Src plays a critical role in US28-mediated signaling and SMC migration.     

The human cytomegalovirus-encoded chemokine receptor US28 induces caspase-dependent apoptosis

Viral subversion of apoptosis regulation plays an important role in the outcome of host/virus interactions. Although human cytomegalovirus (HCMV) encodes several immediate early (IE) antiapoptotic proteins (IE1, IE2, vMIA and vICA), no proapoptotic HCMV protein has yet been identified. Here we show that US28, a functional IE HCMV-encoded chemokine receptor, which may be involved in both viral dissemination and immune evasion, constitutively induces apoptosis in several cell types. In contrast, none of nine human cellular chemokine receptors, belonging to three different subfamilies, induced any significant level of apoptosis. US28-induced cell death involves caspase 10 and caspase 8 activation, but does not depend on the engagement of cell-surface death receptors of the tumour necrosis factor receptor/CD95 family. US28 cell-death induction is prevented by coexpression of C-FLIP, a protein that inhibits Fas-associated death domain protein (FADD)-mediated activation of caspase 10 and caspase 8, and by coexpression of the HCMV antiapoptotic protein IE1. The use of US28 mutants indicated that the DRY sequence of its third transmenbrane domain, required for constitutive G-protein signalling, and the US28 intracellular terminal domain required for constitutive US28 endocytosis, are each partially required for cell-death induction. Thus, in HCMV-infected cells, US28 may function either as a chemokine receptor, a phospholipase C activator, or a proapoptotic factor, depending on expression levels of HCMV and/or cellular antiapoptotic proteins.     

Constitutive signaling of the human cytomegalovirus-encoded chemokine receptor US28

Previously it was shown that the HHV-8-encoded chemokine receptor ORF74 shows considerable agonist-independent, constitutive activity giving rise to oncogenic transformation (Arvanitakis, L., Geras-Raaka, E., Varma, A., Gershengorn, M. C., and Cesarman, E. (1997) Nature 385, 347-350). In this study we report that a second viral-encoded chemokine receptor, the human cytomegalovirus-encoded US28, also efficiently signals in an agonist-independent manner. Transient expression of US28 in COS-7 cells leads to the constitutive activation of phospholipase C and NF-kappaB signaling via G(q/11) protein-dependent pathways. Whereas phospholipase C activation is mediated via Galpha(q/11) subunits, the activation of NF-kappaB strongly depends on betagamma subunits with a preference for the beta(2)gamma(1) dimer. The CC chemokines RANTES (regulated on activation, normal T cell expressed and secreted) and MCP-1 (monocyte chemotactic protein-1) act as neutral antagonists at US28, whereas the CX(3)C chemokine fractalkine acts as a partial inverse agonist with IC(50) values of 1-5 nm. Our data suggest that a high level of constitutive activity might be a more general characteristic of viral G protein-coupled receptors and that human cytomegalovirus might exploit this G protein-coupled receptor property to modulate the homeostasis of infected cells via the early gene product US28.     

G protein-coupled receptor kinase 2 positively regulates epithelial cell migration

Cell migration requires integration of signals arising from both the extracellular matrix and messengers acting through G protein-coupled receptors (GPCRs). We find that increased levels of G protein-coupled receptor kinase 2 (GRK2), a key player in GPCR regulation, potentiate migration of epithelial cells towards fibronectin, whereas such process is decreased in embryonic fibroblasts from hemizygous GRK2 mice or upon knockdown of GRK2 expression. Interestingly, the GRK2 effect on fibronectin-mediated cell migration involves the paracrine/autocrine activation of a sphingosine-1-phosphate (S1P) Gi-coupled GPCR. GRK2 positively modulates the activity of the Rac/PAK/MEK/ERK pathway in response to adhesion and S1P by a mechanism involving the phosphorylation-dependent, dynamic interaction of GRK2 with GIT1, a key scaffolding protein in cell migration processes. Furthermore, decreased GRK2 levels in hemizygous mice result in delayed wound healing rate in vivo, consistent with a physiological role of GRK2 as a regulator of coordinated integrin and GPCR-directed epithelial cell migration.     

Characterization of a G protein-coupled guanylyl cyclase-B receptor from bovine tracheal smooth muscle

A G protein-coupled natriuretic peptide-guanylyl cyclase receptor-B (NPR-B) located in plasma membranes from bovine tracheal smooth muscle shows complex kinetics and regulation. NPR-B was activated by natriuretic peptides (CNP-53 > ANP-28) at the ligand extracellular domain, stimulated by Gq-protein activators, such as mastoparan, and inhibited by Gi-sensitive chloride, interacting at the juxtamembrane domain. The kinase homology domain was evaluated by the ATP inhibition of Mn2+-activated NPR-B, which was partially reversed by mastoparan. The catalytic domain was studied by kinetics of Mn2+/Mg2+ and GTP, and the catalytic effect with GTP analogues with modifications of the /gamma phosphates and ribose moieties. Most NPR-B biochemical properties remained after detergent solubilization but the mastoparan activation and chloride inhibition of NPR-B disappeared. Our results indicate that NPR-B is a highly regulated nano-machinery with domains acting at cross-talk points with other signal transducing cascades initiated by G protein-coupled receptors and affected by intracellular ligands such as chloride, Mn2+, Mg2+, ATP, and GTP.     

G protein-coupled receptor kinase 4gamma interacts with inactive Galpha(s) and Galpha13

G protein-coupled receptors (GPCRs) are regulated by multiple families of kinases including GPCR kinases (GRKs). GRK4 is constitutively active towards GPCRs, and polymorphisms of GRK4γ are linked to hypertension. We examined, through co-immunoprecipitation, the interactions between GRK4γ and the Gα and Gβ subunits of heterotrimeric G proteins. Because GRK4 has been shown to inhibit Gα_s-coupled GPCR signaling and lacks a PH domain, we hypothesized that GRK4γ would interact with active Gα_s, but not Gβ. Surprisingly, GRK4γ preferentially interacts with inactive Gα_s and Gβ to a greater extent than active Gα_s. GRK4γ also interacts with inactive Gα₁₃ and Gβ. Functional studies demonstrate that wild-type GRK4γ, but not kinase-dead GRK4γ, ablates isoproterenol-mediated cAMP production indicating that the kinase domain is responsible for GPCR regulation. This evidence suggests that binding to inactive Gα_s and Gβ may explain the constitutive activity of GRK4γ towards Gα_s-coupled receptors.     

Inhibition of vascular smooth muscle G protein-coupled receptor kinase 2 enhances alpha1D-adrenergic receptor constriction

G protein-coupled receptor kinase 2 (GRK2) is a serine/theorinine kinase that phosphorylates and desensitizes agonist-bound G protein-coupled receptors. GRK2 is increased in expression and activity in lymphocytes and vascular smooth muscle (VSM) in human hypertension and animal models of the disease. Inhibition of GRK2 using the carboxyl-terminal portion of the protein (GRK2ct) has been an effective tool to restore compromised beta-adrenergic receptor (AR) function in heart failure and improve outcome. A well-characterized dysfunction in hypertension is attenuation of betaAR-mediated vasodilation. Therefore, we tested the role of inhibition of GRK2 using GRK2ct or VSM-selective GRK2 gene ablation in a renal artery stenosis model of elevated blood pressure (BP) [the two-kidney, one-clip (2K1C) model]. Use of the 2K1C model resulted in a 30% increase in conscious BP, a threefold increase in plasma norepinephrine levels, and a 50% increase in VSM GRK2 mRNA levels. BP remained increased despite VSM-specific GRK2 inhibition by either GRK2 knockout (GRK2KO) or peptide inhibition (GRK2ct). Although betaAR-mediated dilation in vivo and in situ was enhanced, alpha(1)AR-mediated vasoconstriction was also increased. Further pharmacological experiments using alpha(1)AR antagonists revealed that GRK2 inhibition of expression (GRK2KO) or activity (GRK2ct) enhanced alpha(1D)AR vasoconstriction. This is the first study to suggest that VSM alpha(1D)ARs are a GRK2 substrate in vivo.     

Activation of G protein-coupled estrogen receptor induces endothelium-independent relaxation of coronary artery smooth muscle

Estrogens can either relax or contract arteries via rapid, nongenomic mechanisms involving classic estrogen receptors (ER). In addition to ERα and ERβ, estrogen may also stimulate G protein-coupled estrogen receptor 1 (GPER) in nonvascular tissue; however, a potential role for GPER in coronary arteries is unclear. The purpose of this study was to determine how GPER activity influenced coronary artery reactivity. In vitro isometric force recordings were performed on endothelium-denuded porcine arteries. These studies were augmented by RT-PCR and single-cell patch-clamp experiments. RT-PCR and immunoblot studies confirmed expression of GPER mRNA and protein, respectively, in smooth muscle from either porcine or human coronary arteries. G-1, a selective GPER agonist, produced a concentration-dependent relaxation of endothelium-denuded porcine coronary arteries in vitro. This response was attenuated by G15, a GPER-selective antagonist, or by inhibiting large-conductance calcium-activated potassium (BK(Ca)) channels with iberiotoxin, but not by inhibiting NO signaling. Last, single-channel patch-clamp studies demonstrated that G-1 stimulates BK(Ca) channel activity in intact smooth muscle cells from either porcine or human coronary arteries but had no effect on channels isolated in excised membrane patches. In summary, GPER activation relaxes coronary artery smooth muscle by increasing potassium efflux via BK(Ca) channels and requires an intact cellular signaling mechanism. This novel action of estrogen-like compounds may help clarify some of the controversy surrounding the vascular effects of estrogens.     

G protein-coupled receptor kinase 2 is essential to enable vasoconstrictor-mediated arterial smooth muscle proliferation

Hypertension is associated with increased production and circulation of vasoconstrictors, resulting in enhanced signalling through their cognate G protein-coupled receptors (GPCR). Prolonged vasoconstrictor GPCR signalling increases arterial contraction and stimulates signalling pathways that promote vascular smooth muscle cell (VSMC) proliferation, contributing to the development of atherosclerotic plaques, re-stenosis lesions and vascular remodelling. GPCR signalling through phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) promotes VSMC proliferation. In VSMC, G protein-coupled receptor kinase 2 (GRK2) is known to regulate numerous vasoconstrictor GPCRs and their downstream signalling pathways. As GRK2 is implicated in controlling various aspects of cellular growth, we examined whether GRK2 could affect VSMC proliferation. Using two indices of cell growth, we show that PI3K inhibition and depletion of GRK2 expression produced a similar ablation of pro-proliferative vasoconstrictor-stimulated VSMC growth. Furthermore, GRK2-knockdown ablated the sustained phase of endothelin-1 and angiotensin-II-stimulated Akt phosphorylation, whilst the peak (5 min) phase was unaffected. Conversely, the GRK2 inhibitor compound 101 did not affect vasoconstrictor-driven Akt phosphorylation. Vasoconstrictor-stimulated phosphorylation of the Akt substrates GSK3α and GSK3β was ablated following RNAi-mediated GRK2 depletion, or after PI3K inhibition. Moreover, GRK2 knockdown prevented endothelin-1 and angiotensin-II from increasing cyclin D1 expression.These data suggest GRK2 expression is essential to facilitate vasoconstrictor-driven VSMC proliferation through its ability to promote efficient prolonged PI3K-Akt signalling, and thus relieve the GSK3-mediated block on cell cycling. Considering VSMC GRK2 expression increases early in the development of hypertension, this highlights the potential for GRK2 to promote VSMC growth and exacerbate hypertensive pathophysiological vascular remodelling.     

Identification of novel allosteric nonpeptidergic inhibitors of the human cytomegalovirus-encoded chemokine receptor US28

Human cytomegalovirus (HCMV) is a widespread human pathogen, possessing onco-modulatory properties. Constitutive signaling of the HCMV-encoded chemokine receptor US28 and its ability to bind a broad spectrum of chemokines might facilitate HCMV-associated tumor progression. Novel nonpeptidergic chemotypes were identified as neutral antagonists or inverse agonists on US28, that allosterically inhibit chemokine binding to US28.     

Protein kinase A and G protein-coupled receptor kinase phosphorylation mediates beta-1 adrenergic receptor endocytosis through different pathways

Agonist-induced phosphorylation of beta-adrenergic receptors (beta ARs) by G protein-coupled receptor kinases (GRKs) results in their desensitization followed by internalization. Whether protein kinase A (PKA)-mediated phosphorylation of beta ARs, particularly the beta 1AR subtype, can also trigger internalization is currently not known. To test this, we cloned the mouse wild type beta 1AR (WT beta 1AR) and created 3 mutants lacking, respectively: the putative PKA phosphorylation sites (PKA-beta 1AR), the putative GRK phosphorylation sites (GRK-beta 1AR), and both sets of phosphorylation sites (PKA-/GRK-beta 1AR). Following agonist stimulation, both PKA-beta 1AR and GRK-beta 1AR mutants showed comparable increases in phosphorylation and desensitization. Saturating concentrations of agonist induced only 50% internalization of either mutant compared with wild type, suggesting that both PKA and GRK phosphorylation of the receptor contributed to receptor sequestration in an additive manner. Moreover, in contrast to the WT beta 1AR and PKA-beta 1AR, sequestration of the GRK-beta 1AR and PKA-/GRK-beta 1AR was independent of beta-arrestin recruitment. Importantly, clathrin inhibitors abolished agonist-dependent internalization for both the WT beta 1AR and PKA-beta 1AR, whereas caveolae inhibitors prevented internalization only of the GRK-beta 1AR mutant. Taken together, these data demonstrate that: 1) PKA-mediated phosphorylation can trigger agonist-induced internalization of the beta 1AR and 2) the pathway selected for beta 1AR internalization is primarily determined by the kinase that phosphorylates the receptor, i.e. PKA-mediated phosphorylation directs internalization via a caveolae pathway, whereas GRK-mediated phosphorylation directs it through clathrin-coated pits.     

G protein-coupled receptor kinase interaction with Hsp90 mediates kinase maturation

G protein-coupled receptor kinase 2 (GRK2) is a serine/threonine-specific protein kinase that mediates agonist-dependent phosphorylation of numerous G protein-coupled receptors. In an effort to identify proteins that regulate GRK2 function, we searched for interacting proteins by immunoprecipitation of endogenous GRK2 from HL60 cells. Subsequent analysis by gel electrophoresis and mass spectrometry revealed that GRK2 associates with heat shock protein 90 (Hsp90). GRK2 interaction with Hsp90 was confirmed by co-immunoprecipitation and was effectively disrupted by geldanamycin, an Hsp90-specific inhibitor. Interestingly, geldanamycin treatment of HL60 cells decreased the expression of endogenous GRK2 in a dose- and time-dependent manner, and metabolic labeling demonstrated that geldanamycin rapidly accelerated the degradation of newly synthesized GRK2. The use of various protease inhibitors suggested that GRK2 degradation induced by geldanamycin was predominantly through the proteasome pathway. To test whether Hsp90 plays a general role in regulating GRK maturation, additional GRKs were studied by transient expression in COS-1 cells and subsequent treatment with geldanamycin. These studies demonstrate that GRK3, GRK5, and GRK6 are also stabilized by interaction with Hsp90. Taken together, our work revealed that GRK interaction with heat shock proteins plays an important role in regulating GRK maturation.     

Galpha13 stimulates cell migration through cortactin-interacting protein Hax-1

Galpha13, the alpha-subunit of the heterotrimeric G protein G13, has been shown to stimulate cell migration in addition to inducing oncogenic transformation. Cta, a Drosophila ortholog of G13, has been shown to be critical for cell migration leading to the ventral furrow formation in Drosophila embryos. Loss of Galpha13 has been shown to disrupt cell migration associated with angiogenesis in developing mouse embryos. Whereas these observations point to the vital role of G13-orthologs in regulating cell migration, widely across the species barrier, the mechanism by which Galpha13 couples to cytoskeleton and cell migration is largely unknown. Here we show that Galpha13 physically interacts with Hax-1, a cytoskeleton-associated, cortactin-interacting intracellular protein, and this interaction is required for Galpha13-stimulated cell migration. Hax-1 interaction is specific to Galpha13, and this interaction is more pronounced with the mutationally or functionally activated form of Galpha13 as compared with the wild-type Galpha13. Expression of Hax-1 reduces the formation of actin stress fibers and focal adhesion complexes in Galpha13-expressing NIH3T3 cells. Coexpression of Hax-1 also attenuates Galpha(13)-stimulated activity of Rho while potentiating Galpha13-stimulated activity of Rac. The presence of a quadnary complex consisting of Galpha13, Hax-1, Rac, and cortactin indicates the role of Hax-1 in tethering Galpha13 to the cytoskeletal component(s) involved in cell movement. Whereas the expression of Hax-1 potentiates Galpha13-mediated cell movement, silencing of endogenous Hax-1 with Hax-1-specific small interfering RNAs drastically reduces Galpha13-mediated cell migration. These findings, along with the observation that Hax-1 is overexpressed in metastatic tumors and tumor cell lines, suggest a novel role for the association of oncogenic Galpha13 and Hax-1 in tumor metastasis.     

Signaling and regulation of G protein-coupled receptors in airway smooth muscle

Signaling through G protein-coupled receptors (GPCRs) mediates numerous airway smooth muscle (ASM) functions including contraction, growth, and "synthetic" functions that orchestrate airway inflammation and promote remodeling of airway architecture. In this review we provide a comprehensive overview of the GPCRs that have been identified in ASM cells, and discuss the extent to which signaling via these GPCRs has been characterized and linked to distinct ASM functions. In addition, we examine the role of GPCR signaling and its regulation in asthma and asthma treatment, and suggest an integrative model whereby an imbalance of GPCR-derived signals in ASM cells contributes to the asthmatic state.     

Selective regulation of H1 histamine receptor signaling by G protein-coupled receptor kinase 2 in uterine smooth muscle cells

Histamine stimulates uterine contraction; however, little is known regarding the mechanism or regulation of uterine histamine receptor signaling. Here we investigated the regulation of Galpha(q/11)-coupled histamine receptor signaling in human myometrial smooth muscle cells using the inositol 1,4,5-trisphosphate biosensor pleckstrin homology domain of phospholipase-delta1 tagged to enhanced green fluorescent protein and the Ca(2+)-sensitive dye Fluo-4. Histamine addition caused concentration-dependent increases in inositol 1,4,5-trisphosphate and [Ca(2+)](i) in the ULTR human uterine smooth muscle cell line and primary human myometrial cells. These effects were completely inhibited by the H(1) histamine receptor antagonist, diphenhydramine, and were unaffected by the H(2) histamine receptor antagonist, cimetidine. ULTR and primary myometrial cells were transfected with either dominant-negative G protein-coupled receptor kinases (GRKs) or small interfering RNAs targeting specific GRKs to assess the roles of this protein kinase family in H(1) histamine receptor desensitization. Dominant-negative GRK2, but not GRK5 or GRK6, prevented H(1) histamine receptor desensitization. Similarly, transfection with short interfering RNAs (that each caused >70% depletion of the targeted GRK) for GRK2, but not GRK3 or GRK6, also prevented H(1) histamine receptor desensitization. Our data suggest that histamine stimulates phospholipase C-signaling in myometrial smooth muscle cells through H(1) histamine receptors and that GRK2 recruitment is a key mechanism in the regulation of H(1) histamine receptor signaling in human uterine smooth muscle. These data provide insights into the in situ regulation of this receptor subtype and may inform pathophysiological functioning in preterm labor and other conditions involving uterine smooth muscle dysregulation.