The N Terminus of Kaposi's Sarcoma-associated Herpesvirus G Protein-coupled Receptor Is Necessary for High Affinity Chemokine Binding but Not for Constitutive Activity

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) contains a gene encoding a G protein-coupled receptor (KSHV-GPCR) that is homologous to mammalian chemokine receptors. KSHV-GPCR signals constitutively (in an agonist-independent manner) via the phosphoinositide-inositol 1,4,5-trisphosphate pathway. Because it has been proposed that the N terminus (N-TERM) of other GPCRs may act as tethered agonists, we determined whether the N-TERM of KSHV-GPCR is necessary for constitutive signaling activity or ligand binding, or both. We show that replacement of the entire N-TERM of KSHV-GPCR with those of two other GPCRs, deletion of residues within the N-TERM, and disruption of a putative disulfide bond that may hold the N-TERM in close proximity to extracellular loop 3 do not affect constitutive signaling activity but decrease chemokine binding. There were differences in the effects of mutation of the N-TERM on binding of the chemokines growth-related oncogene ␣, which is an agonist, and interferon-␥-inducible protein-10, which is an inverse agonist. The effects on chemokine binding were accompanied by changes in chemokine regulation of KSHV-GPCR signaling. We conclude that the N-TERM is not necessary for constitutive KSHV-GPCR signaling, i.e. the N-TERM is not a tethered agonist, but plays a crucial role in binding of chemokine ligands and of chemokine regulation of KSHV-GPCR signaling.

Virally encoded 7TM receptors

“…Through protein engineering of ORF74 a series of pharmacological phenotypes with modulated constitutive activity and/or ligand binding pro®le have been generated, which can be exploited to address this basic question. For example, truncations of the N-terminus of ORF74 eliminates binding of all chemokines, without in¯uencing its high basal activity as presented in Figure 4, upper left box (Ho et al, 1999;. Another interesting phenotype in which the agonist ± but not inverse agonist ± action is eliminated, is obtained by mutating the Arg residues at the extracellular ends of TM-V (Figure 4, upper right box).…”

Kaposi's Sarcoma-associated Herpesvirus G Protein-coupled Receptor Signals through Multiple Pathways in Endothelial Cells

“…In order to obtain clonal transfectants, selected neomycin plates were split to obtain isolated colonies. The clones and pools (containing multiple clones) were screened for KSHV-GPCR expression by measuring specific binding with radiolabeled growth-related oncogene ␣ (Gro␣), a ligand for KSHV-GPCR (15,16), and receptor signaling through the inositol phosphate (InsP) pathway. One pool and one clone expressing KSHV-GPCRs (MLEC/KSHV-GPCR), and one pool transfected with empty plasmid (MLEC/mock) were chosen for further studies.…”

Charged Residues at the Intracellular Boundary of Transmembrane Helices 2 and 3 Independently Affect Constitutive Activity of Kaposi's Sarcoma-associated Herpesvirus G Protein-coupled Receptor

“…It seems, however, that the ability of the inverse agonists to inhibit signaling by these receptors was inversely related to the level of constitutive activity of the receptor. Other KSHV-GPCR mutants have been found to be unresponsive to inverse agonist inhibition but these were receptors with mutations in their amino-terminal domains that caused loss of binding (10,11).…”

“…In a previous report (10), we tested the hypothesis that the amino-terminal extracellular domain of KSHV-GPCR may serve as a "tethered ligand" that constitutively activates the receptor. Although we found that the amino terminus was important for high affinity binding of chemokines, we showed that the amino terminus was not important for constitutive signaling.…”