Identification of the First Nonpeptidergic Inverse Agonist for a Constitutively Active Viral-encoded G Protein-coupled Receptor

Abstract

Human cytomegalovirus (HCMV) encodes a G proteincoupled receptor (GPCR), named US28, which shows homology to chemokine receptors and binds several chemokines with high affinity. US28 induces migration of smooth muscle cells, a feature essential for the development of atherosclerosis, and may serve as a co-receptor for human immunodeficiency virus-type 1 entry into cells. Previously, we have shown that HCMV-encoded US28 displays constitutive activity, whereas its mammalian homologs do not. In this study we have identified a small nonpeptidergic molecule (VUF2274) that inhibits US28-mediated phospholipase C activation in transiently transfected COS-7 cells and in HCMV-infected fibroblasts. Moreover, VUF2274 inhibits US28-mediated HIV entry into cells. In addition, VUF2274 fully displaces radiolabeled RANTES (regulated on activation normal T cell expressed and secreted) binding at US28, apparently with a noncompetitive behavior. Different analogues of VUF2274 have been synthesized and pharmacologically characterized, to understand which features are important for its inverse agonistic activity. Finally, by means of mutational analysis of US28, we have identified a glutamic acid in transmembrane 7 (TM 7), which is highly conserved among chemokine receptors, as a critical residue for VUF2274 binding to US28. The identification of a full inverse agonist provides an important tool to investigate the relevance of US28 constitutive activity in viral pathogenesis.

Constitutive Signaling of the Human Cytomegalovirus-encoded Receptor UL33 Differs from That of Its Rat Cytomegalovirus Homolog R33 by Promiscuous Activation of G Proteins of the Gq, Gi, and Gs Classes

“…Our current data and those reported earlier by us and others (8,9,10,16,17) indicate that CMV effectively uses vGPCRs to orchestrate signaling networks within the cell during its viral life span. Previously, four genes encoding vGPCRs have been identified in the HCMV genome (US27, US28, UL33, and UL78) (4).…”

“…Previously, US28 has been shown to constitutively activate a variety of signal transduction cascades, including PLC (8), MAPK pathways (41), and various transcription factors (8,17). Moreover, we and others have recently shown that also in HCMV-infected fibroblasts US28 constitutively activates PLC (9,10), further emphasizing the physiological relevance of the constitutive signaling of US28 after viral infection. Here, we show that another HCMV-encoded receptor, UL33, displays constitutive signaling in transfected as well as in HCMV-infected cells.…”

“…Similarly, the CC chemokines RANTES (regulated on activation normal T cell expressed and secreted), and MIP-1␣ did not modulate UL33 constitutive activity nor showed any binding to this receptor (data not shown). The recent identification of a small non-peptidergic inverse agonist for US28 by its modulating effect on US28-mediated constitutive signaling (9), demonstrates that the UL33-mediated signaling assays described in this study may be suitable screening systems for the identification of ligands acting at UL33. The availability of such a ligand would be of help in elucidating the role of UL33 in HCMV infection.…”

Constitutively active Gq/11-coupled Receptors Enable Signaling by Co-expressed Gi/o-coupled Receptors

“…The gifts of acrivastine (The Wellcome Foundation Ltd.), GR-127935 (Dr. P. R. Saxena), mianserin hydrochloride (Organon N.V., Oss, The Netherlands), (R)-and (S)-cetirizine hydrochloride (UCB Pharma), SB-224289 (SmithKline Beecham), and pcDEF 3 (Dr. J. Langer) and of the cDNAs encoding bovine G␥ 2 (Dr. M. Lohse), G␤ 2 (Dr. I. Iyengar), Clostridium botulinum c 3 exoenzyme (Dr. S. Narumiya), US28 (encoded by VHL/E HCMV strain, GenBank TM accession number L20501, bases 219000 -220263) in pcDNA 3 (Dr R. Doms), the human CCR1 receptor in pcDNA 3 (Dr. C. Tensen), the human serotonin 5-HT 1B receptor in pKCREH (Dr. N. Stam), the human muscarinic M 2 receptor in pcD (Dr. R. Maggio), the human adenosine A 1 receptor in pcDNA 3 DNA Constructs-⌬(2-22)-US28 and the HA-tagged versions of both wild-type and ⌬(2-22)-US28 were generated by PCR as described previously (18). The single amino acid mutation for US28-R129A was introduced using the Altered Sites® II in vitro mutagenesis system (Promega, Madison, WI) as described earlier (22).…”

“…Various polymorphic GPCR variants have been shown to be highly constitutively active (15,16), but also certain wild-type GPCRs, such as the histamine H 3 receptor, exhibit high constitutive activity in vivo (17). Moreover, viral infection of cells may also result in the expression of virally encoded GPCRs that exhibit high levels of constitutive activity (18,19).…”