kshv - gpcrs were expressed   in cos-1 and nih 3t3 cells by transfection with pckshv - gpcr ( 6 ) or pcefl - kshv - gpcr ( 7 ) using the protocols described previously .
the c - x - c chemokines huip-10 and humig , the mouse homologues muip-10 and mumig , and the   huip-10 analogues were chemically synthesized using established   procedures ( 12 ) .
cos-1 cells transiently transfected with plasmid encoding   kshv - gpcr ( 5 g / ml ) or nih 3t3 cells stably expressing   kshv - gpcrs were labeled with myo-[h]inositol , and the formation of [ h]inositol phosphates during a 90-min incubation   was measured as described ( 6 , 7 ) .
the full - length kshv - gpcr cdna in pcdna3.1(+ ) ( pckshv - gpcr ; reference 6 ) was used for mutation .
mutants were prepared by pcr and were subcloned directly into pckshv - gpcr after digesting with ecori and   ecorv .
kshv - gpcr  expressing nih 3t3   cells were grown in dme supplemented with 10% calf serum .
dna   synthesis was measured by incubating kshv - gpcr  expressing   nih 3t3 cells in medium supplemented with 1% calf serum and   the indicated concentration of huip-10 for 2 d before incubation   with [ h]thymidine ( 2 ci / ml ) for 46 h. statistical significance was determined in   each experiment by student 's t test .
kshv - gpcrs were expressed   in cos-1 and nih 3t3 cells by transfection with pckshv - gpcr ( 6 ) or pcefl - kshv - gpcr ( 7 ) using the protocols described previously .
the c - x - c chemokines huip-10 and humig , the mouse homologues muip-10 and mumig , and the   huip-10 analogues were chemically synthesized using established   procedures ( 12 ) .
cos-1 cells transiently transfected with plasmid encoding   kshv - gpcr ( 5 g / ml ) or nih 3t3 cells stably expressing   kshv - gpcrs were labeled with myo-[h]inositol , and the formation of [ h]inositol phosphates during a 90-min incubation   was measured as described ( 6 , 7 ) .
the full - length kshv - gpcr cdna in pcdna3.1(+ ) ( pckshv - gpcr ; reference 6 ) was used for mutation .
mutants were prepared by pcr and were subcloned directly into pckshv - gpcr after digesting with ecori and   ecorv .
kshv - gpcr  expressing nih 3t3   cells were grown in dme supplemented with 10% calf serum .
dna   synthesis was measured by incubating kshv - gpcr  expressing   nih 3t3 cells in medium supplemented with 1% calf serum and   the indicated concentration of huip-10 for 2 d before incubation   with [ h]thymidine ( 2 ci / ml ) for 46 h.
1 illustrates that huip-10 specifically inhibits inositol phosphate second messenger formation in cos-1 cells   expressing kshv - gpcrs .
the dose of huip-10 that   causes 50% inhibition of inositol phosphate formation is 15   nm .
huip-10 had no effect on inositol phosphate production by cos-1 cells that did not express kshv - gpcrs   ( data not shown ) .
these data are consistent with the idea   that huip-10 is a specific inverse agonist ( or negative antagonist ; reference 13 ) of kshv - gpcr .  
as noted above , huip-10 is a ligand for cxcr3 , which   also binds humig ( 10 ) .
to further characterize the effects of these   chemokines on kshv - gpcr signaling , the effects of humig and of the mouse homologues muip-10 and mumig   were determined .
muip-10 , like huip-10 , inhibits signaling by kshv - gpcr , whereas humig and mumig have   no effect on kshv - gpcr signaling ( fig .
the   ip-10 homologues are inverse agonists , whereas the mig homologues exhibit no detectable activity at kshv - gpcr .
huip-10 is a 77amino acid polypeptide . to gain insight   into the structural domain of huip-10 involved in inhibition   of kshv - gpcr signaling ,
huip-10(477 ) and huip-10(977 ) are lacking the first three or eight amino acid residues , respectively , of huip-10 .
both huip-10(477 ) and   huip-10(977 ) inhibited kshv - gpcr signaling although   with lower potencies than huip-10 ( 3- and 10-fold lower ,   respectively ; fig .
2 a ) . thus , the nh2 terminus of huip-10   is not needed for inhibition of kshv - gpcr signaling .
this   is an interesting finding because the agonist activity of several   members of the c - x - c chemokine family , including huip-10 , has been shown to be dependent on their nh2-terminal   domains ( 8) .
for example , deletion of the nh2 terminus of   il-8 causes loss of agonist activity and forms peptides that act   as competitive antagonists ( 15 ) .
the active domain ( or pharmacophore ) for inhibition of kshv - gpcr signaling is not   part of the nh2 terminus of huip-10 .   it has been found that the putative extracellular nh2 termini of cxcr1 and cxcr2 are important for interacting   with il-8 ( 16 ) . therefore , we constructed a mutant kshv - gpcr in which amino acid residues from positions 211   were deleted [ kshv - gpcr(211 ) ] .
therefore ,   residues within the nh2 terminus may affect constitutive   signaling by kshv - gpcr .
however , since signaling activity is directly related to receptor number ( 6 ) , it is possible   that the modest decrease in signaling is due to decreased   expression .
more importantly , the constitutive signaling   activity exhibited by kshv - gpcr(211 ) was not inhibited by huip-10 .
as we have not been able to measure   specific huip-10 binding , perhaps because of binding to   cell surface heparin sulfates ( 17 ) , we do not know whether   this lack of effect of huip-10 is because it does not bind to   kshv - gpcr(211 ) or whether it binds but does not   inactivate kshv - gpcr(211 ) .
however , it is clear that   the nh2 terminus of kshv - gpcr is important for the inverse agonist action of huip-10 .
fibroblasts may be a good model for the study of   kshv - gpcr function because fibroblasts are typically   present in ks lesions ( 1 ) and kshv - like viruses are found   in retroperitoneal fibromatosis tissues in monkeys ( 18 ) .
expression of kshv - gpcr results in stimulation of proliferation of nih 3t3 fibroblasts ( 7 ) .
stimulation of the proliferation of target cells by kshv - gpcr could be a component   of the pathogenesis of ks or primary effusion lymphomas   ( 1 , 2 ) . to determine whether inhibition of kshv - gpcr   signaling would inhibit the proliferative response , the effects of the ip-10 homologues were measured in transfected nih 3t3 cells stably expressing kshv - gpcrs .  
3 ( top ) illustrates that huip-10 and muip-10 inhibit   kshv - gpcr signaling in nih 3t3 cells , whereas humig and mumig have no effect .
moreover , huip-10 causes   dose - dependent inhibitions of kshv - gpcr signaling and   stimulated dna synthesis in these cells ( fig .
the concentrations required for 50% inhibition ( ic50s ) of   these effects are similar : 39 nm for inhibition of inositol   phosphate second messenger formation , and 29 nm for inhibition of dna synthesis .
these results indicate that   huip-10 is an inverse agonist of constitutive signaling by   kshv - gpcr in nih 3t3 cells that inhibits kshv - gpcr  stimulated cell proliferation .  
ip-10 homologues are cxcr3 agonists ( 9 ) , but we   show herein that they are inverse agonists at kshv - gpcr . in the majority of instances where two gpcrs interact with the same ligand , the receptors respond similarly   to that ligand ( 19 ) . however , there are examples of mutations of gpcrs engineered in the laboratory that have   changed an antagonist or inverse agonist into a partial agonist ( 20 , 21 ) .
the different effects of the ip-10 homologues   at kshv - gpcr and cxcr3 may represent a related circumstance in which a pirated mammalian receptor is mutated within a virus and is then antagonized by a naturally   occurring agonist .
kshv - gpcr interacts with a much broader array of   chemokines ( 6 ) than most mammalian receptors ( 8) . indeed , kshv - gpcr interacts with c - x - c chemokines including il-8 , neutrophil activating protein 2 , platelet factor   4 , melanoma growth stimulatory activity , ip-10 analogues ,   and c - c chemokines including regulated on activation ,   normal t expressed and secreted ( rantes ) and i-309 .  
however , of these chemokines only the ip-10 analogues   inhibit kshv - gpcr signaling .
it is noteworthy that in addition to encoding kshv - gpcr , there are three chemokine analogues encoded within the kshv genome .
one of   these , vmip - ii , is a broad - spectrum antagonist of chemokine receptors ( 22 ) , but stimulates chemotaxis of eosinophils via chemokine receptor ccr3 ( 23 ) .
vmip - i and   vmip - ii block infection by hiv-1 by binding to chemokine receptors ccr3 and ccr5 , and stimulate angiogenesis ( 23 ) .
it is not known whether any of these chemokines   affects kshv - gpcr signaling .
evidence continues to accumulate supporting the idea   that kshv is involved in the pathogenesis of ks ( 1 ) and   primary effusion lymphomas ( 2 ) .
more recently , it has been   proposed that kshv could also be involved in the development of multiple myeloma ( 24 ) .
from the perspective of   demonstrating a pathogenic role for kshv - gpcr in   these diseases , it will be important to develop an animal   model and to show that inhibition of kshv - gpcr signaling inhibits tumorigenesis in kshv - infected animals .  
the discovery of an inhibitor of the constitutive signaling   activity of kshv - gpcr will allow direct testing in an animal model of the hypothesis that kshv infection leads to   expression of kshv - gpcr and kshv - gpcr signaling   leads to tumorigenesis .
effects of huip-10   ( filled squares ) , humig ( filled circles ) , muip-10 ( open squares ) , and   mumig ( open circles ) on constitutive kshv - gpcr signaling .  
inositol phosphate accumulation   in cos-1 cells transiently transfected with plasmid encoding   kshv - gpcr was measured as   described in materials and methods .
chemokine analogues were   added at the concentrations indicated 15 min before licl ( 10 mm ) .
untransfected cells and cells transfected with plasmid without dna encoding kshv - gpcr (  mock  ) or with plasmid encoding the receptor for   thyrotropin - releasing hormone were studied in parallel .
there was no effect of any of these chemokines on inositol phosphate production in untransfected cells , in mock - transfected cells , or in cells expressing thyrotropin - releasing hormone receptors ( not shown ) .
the dose of huip-10 that   caused 50% inhibition of inositol phosphate formation was 15 nm ( 7.232   nm , 95% confidence interval ) .
the data represent the mean  sd of triplicate determinations in a representative of three experiments .
effects of truncations of the nh2 termini of   huip-10 and kshv - gpcr on   inhibition of kshv - gpcr signaling .
inositol phosphate accumulation in cos-1 cells transiently transfected with plasmid   encoding kshv - gpcr or   kshv - gpcr(211 ) was measured as described in materials   and methods .
huip-10 , huip-10(477 ) , or huip-10(977 ) was   added at the concentrations indicated 15 min before licl ( 10 mm ) .  
( a ) effect of truncating the nh2   terminus of the ligand huip-10 .  
the data represent the mean  sd   of triplicate determinations in a   representative of three experiments . filled squares , huip-10 ;   open circles , huip-10(477 ) ; filled   triangles , huip-10(977 ) .
the data represent   the mean  sd of triplicate determinations in a representative experiment in   which two plasmid clones encoding kshv - gpcr(211 ) were tested .  
inhibition of constitutive kshv - gpcr signaling   and dna synthesis in nih 3t3   mouse fibroblasts .
( a ) inositol   phosphate accumulation in nih   3t3 cells stably expressing   kshv - gpcrs ( reference 7 ) .  
huip-10 , humig , muip-10 , or   mumig ( 300 m ) was added 15   min before the addition of licl   ( 10 mm ) .
the data represent the   mean  sd of triplicate determinations in one of two experiments .
( b ) dose - dependent effects of huip-10 on inositol   phosphate formation ( open circles )   and dna synthesis ( filled   squares ) .
the concentration of   huip-10 required for 50% inhibition ( ic50 ) of inositol phosphate second messenger formation is 39  nm ( 9160 nm ; 95%   confidence interval ) , and for 50% inhibition of dna synthesis is 29 nm   ( 8110 nm , 95% confidence interval ) .
 a g protein  coupled receptor ( gpcr ) is encoded within the genome of kaposi 's sarcoma associated herpesvirus ( kshv)/human herpesvirus 8 , a virus that may be involved in the pathogenesis of kaposi 's sarcoma and primary effusion lymphomas . 
 kshv - gpcr exhibits constitutive signaling activity that causes oncogenic transformation . 
 we report that human interferon   ( ifn)-inducible protein 10 ( huip-10 ) , a c - x - c chemokine , specifically inhibits signaling of   kshv - gpcr . 
 in contrast , monokine induced by ifn- ( humig ) , which like huip-10 is an   agonist of c - x - c chemokine receptor 3 , does not inhibit kshv - gpcr signaling . 
 moreover ,   huip-10 , but not humig , inhibits kshv - gpcr  induced proliferation of nih 3t3 cells .   
 these results show that huip-10 is an inverse agonist that converts kshv - gpcr from an active to an inactive state . 
 thus , a human chemokine inhibits constitutive signaling and cellular   proliferation that is mediated by a receptor encoded by a human disease - associated herpesvirus . 
Materials and Methods
Inositol Phosphate Accumulation.
Mutagenesis.
Cell Proliferation Assays.
Statistical Analysis.
Results and Discussion
Figures and Tables