cos-1 cells were transfected by the deae - dextran method   with plasmid encoding kshv - gpcr ( pckshv - gpcr ) as described ( 7 ) . in different experiments , plasmids encoding grk2 ,   4 , 5 , or 6 , cxcr2 , or g16 were transfected . all transfection cocktails were made to contain equal amounts of plasmid dna by the   addition of appropriate amounts of vector . 24 h after transfection ,   cells were harvested and reseeded in 12-well plates in dulbecco 's   modified eagle 's medium with 10% nuserum and 1 ci / ml myo- [ h]inositol ( where indicated ) , and maintained in culture in 5%   co2 at 37c for an additional 24 h. after removing the culture   medium and washing with buffer , cells were incubated in hank 's   balanced salt solution containing 25 mm hepes , ph 7.4 , and 10   mm licl . cells were harvested after 90 min and ip accumulation   was measured using ion exchange chromatography as described   previously ( 20 ) . ip accumulation was measured as the h - radioactivity in ips as a fraction of the h - radioactivity in inositol lipids   plus ips ( lipids + ips ) . , cos-1 cells were transfected with plasmid encoding   kshv - gpcr ( 10 g / ml ) in the absence or presence of plasmid   encoding grk5 ( 3 g / ml ) and tested for il-8 binding 48 h after   transfection . approximately 100,000 cells / well were washed with   hank 's balanced salt solution containing 25 mm hepes , ph 7.4 .   between 0.3 and 0.5 nm il-8 in the absence or presence of   various concentrations of unlabeled il-8 was added and binding   was allowed to proceed at 4c for 2 h. the binding buffer contained bovine serum albumin ( 1 mg / ml ) , bacitracin ( 1 mg / ml ) ,   and phenyl methyl sulfonyl fluoride ( 1 mm ) . assays were terminated by aspirating the binding buffer , washing three times with   chilled buffer , and solubilizing the cells with 0.4 n naoh . an   aliquot of this solution was counted in a gamma counter and the   data were expressed as cpm bound . binding curves were analyzed   using prism software ( graphpad , inc . , san diego , ca ) . for experiments with nih 3t3 cells or nrk cells , the cdna   encoding kshv - gpcr was subcloned into plasmid pcefl ( a   gift from dr . silvio gutkind , national institute of dental research ,   national institutes of health ) . nih 3t3 cells and nrk cells were   transfected by the calcium phosphate method with plasmid encoding kshv - gpcr ( pceflkshv - gpcr ) or with pcefl   without or with plasmids encoding grk2 , 4 , 5 , or 6 . on the day   after transfection , the medium was removed and the cells were   incubated in dulbecco 's modified eagle 's medium with 10% calf   serum and 0.75 mg / ml geneticin . plasmids encoding bovine grk2 , human grk4 ,   bovine grk5 , and human grk6 were provided by dr . robert   j. lefkowitz ( howard hughes medical institute , duke university medical center , durham , nc ) . cos-1 and   nih 3t3 cells were from the american type culture collection   ( rockville , md ) . myo-[h]inositol and i  il-8 were purchased   from dupont new england nuclear ( boston , ma ) . dulbecco 's   modified eagle 's medium , geneticin , calf serum , and hank 's balanced salt solution were from gibco brl ( gaithersburg , md ) . plasmids encoding bovine grk2 , human grk4 ,   bovine grk5 , and human grk6 were provided by dr . robert   j. lefkowitz ( howard hughes medical institute , duke university medical center , durham , nc ) . cos-1 and   nih 3t3 cells were from the american type culture collection   ( rockville , md ) . myo-[h]inositol and i  il-8 were purchased   from dupont new england nuclear ( boston , ma ) . dulbecco 's   modified eagle 's medium , geneticin , calf serum , and hank 's balanced salt solution were from gibco brl ( gaithersburg , md ) . to determine whether constitutive signaling by kshv - gpcrs could be inhibited by grks , we coexpressed   kshv - gpcr and grk2 , 4 , 5 , or 6 in cos-1 cells expression of   grk2 , 4 , 5 , or 6 alone had no effect on ip formation ( data   not shown ) . fig . 1 a illustrates that grks 4 , 5 , and 6 , but   not grk2 , inhibited kshv - gpcr stimulation of ip formation by 4163% . the lack of effect of grk2 on kshv - gpcr signaling was not due to lack of its expression because grk2 inhibited agonist - stimulated signaling by the   thyrotropin - releasing hormone receptor by 20  9.4% in   parallel incubations . of note , grks 4 , 5 , and 6 appear to   comprise a distinct subfamily of receptor - specific kinases ( 14 ) . specificity of desensitization by different grks has been   observed ( 21 , 22 ) . b confirms that grk5 inhibits   kshv - gpcr signaling and shows that the extent of inhibition of ip formation by grk5 was similar , 53% , at all   levels of kshv - gpcr expression . this level of inhibition   is similar to that found for inhibition of agonist - stimulated   ip second messenger formation when 1b - adrenergic receptors and grks were coexpressed in cos-7 cells ( 22 ) .   to determine whether the effect of grk5 to inhibit ip   formation was general or whether it may be specific for   kshv - gpcr , we coexpressed grk5 and cxcr2 . because chemokine signaling via cxcr2 in cos cells requires g16 ( or g14 or g15 ) coexpression of g16 does not enhance kshv - gpcr   signaling in cos-1 cells ( 7 ) . we reasoned that it may be   necessary to activate cxcr2 before measuring the effect   of the grk on agonist - stimulated signaling so as to have   activated cxcr2 as a substrate for grk - mediated phosphorylation . therefore , we measured the effects of grk5   on il-8 stimulation of ip formation via cxcr2 when il-8   was added acutely or added for 1 or 48 h before ip measurement . as we showed previously ( 7 ) , il-8 stimulated ip   formation via cxcr2 to a level only 5565% of that stimulated constitutively by kshv - gpcr under these conditions . in this series of experiments , kshv - gpcr stimulation of ip formation in contrast , grk5 had no effect on il-8   stimulation of ip formation via cxcr2 whether il-8 was   present only during the period of ip measurement or chronically . thus , it appears that grk5 does not desensitize all   gpcrs under these conditions , but there is specificity to   the effect of grk5 on kshv - gpcr signaling . it was recently proposed that grks ( and arrestin ) are involved in gpcr internalization ( 23 ) . because enhanced   rates of internalization can lead to increased rates of receptor degradation ( 24 ) , it was possible that the decreased signaling observed when kshv - gpcr and grk5 were coexpressed was secondary to decreased receptor levels due to   increased receptor turnover . decreased receptor expression   might have occurred by other mechanisms also . we found   that the levels ( and affinities , equilibrium inhibitory constants of 35  12 nm ) of kshv - gpcrs were not different   in cos-1 cells expressing kshv - gpcr alone or kshv - gpcr and grk5 ( data not shown ) . thus , grk inhibition of kshv - gpcr signaling occurred without a change   in kshv - gpcr expression . we considered the possibility that kshv - gpcr signaling could be inhibited by pkc also . 3 illustrates that   phorbol 12-myristate 13-acetate ( pma ) causes a dose - dependent inhibition of ip formation by kshv - gpcr . only active phorbol esters , pma ( 0.1 m , to 36  4.7% of control ) , and phorbol 12,13-didecanoate ( 0.1 m , to 66  5.4%   of control ) , but not an inactive phorbol ester , 4-phorbol   12,13-didecanoate ( 0.1 m , to 96  5.5% of control ) , inhibited kshv - gpcr  stimulated ip formation . these effects occur exclusively via activation of pkc because they   are prevented by a specific pkc inhibitor , calphostin c   ( 25 ; data not shown ) . as was found for cxcr1 ( 19 ) , we   found that pma inhibited il-8 stimulation of ip formation   via cxcr2 ( data not shown ) . thus , kshv - gpcr signaling , like signaling by cxcr1 and cxcr2 , is inhibited   by pkc activation . 4 illustrates that the effects of expression of grk4 ,   5 , or 6 and pkc activation on constitutive ip formation   stimulated by kshv - gpcr are additive . grk2 had no   effect on kshv - gpcr  stimulated ip formation and did   not affect inhibiton by pma . in this series of experiments ,   grk4 , 5 , or 6 inhibited ip formation by 5669% , pma   inhibited ip formation by 65% , and the combination of expression of grk4 , 5 , or 6 and activation of pkc by pma   inhibited ip formation by 8590% .   to show that grk - mediated inhibition of kshv - gpcr   signaling was not peculiar to cos cells in which proteins   may be markedly overexpressed , and that these protein kinases would inhibit an important cellular response to kshv - gpcr , we constructed nih 3t3 and nrk cell populations expressing kshv - gpcrs , kshv - gpcrs and grk2 ,   or kshv - gpcrs and grk5 . mouse nih 3t3 cells and   rat nrk cells are good models because proliferating fibroblasts are typically present in ks lesions ( 4 ) and kshv - like   viruses have recently been found in retroperitoneal fibromatosis tissues in monkeys ( 26 ) . 5 shows that , as in   cos-1 cells , expression of grk5 inhibited ip accumulation in nih 3t3 cells ; grk2 had no effect . more importantly , expression of grk5 , but not grk2 , inhibited proliferation of nih 3t3 cells ( fig . 5 ) . grk4 , 5 , and 6 , but   not grk2 , inhibited kshv - gpcr  stimulated proliferation of nrk cells by > 75% ( data not shown ) . thus , inhibition of kshv - gpcr signaling by expression of grk5   was found in cos-1 and nih 3t3 cells and of kshv - gpcr stimulation of proliferation in nih 3t3 and nrk   cells . we recently showed that expression of kshv - gpcr induces transformation of nih 3t3 cells and that   transformation is inhibited by coexpression of grk5 but   not of grk2 ( 8) . more commonly , however ,   agonist - independent signaling has been observed with mutated gpcrs including a number that have been associated   with human diseases ( 30 ) , including nonmalignant tumors   ( 31 , 32 ) . moreover , gpcrs have been found to be oncogenic under some circumstances ( 33 ) . although herpesvirus   saimiri is an oncogenic virus ( 1 ) , it is not known whether   the gpcr encoded within the genome of this virus is involved in the transformation process . as kshv is found in   primary effusion lymphomas and ks tissues ( 3 , 4 , 34 ) and   kshv - gpcr stimulates proliferation of fibroblasts in tissue culture ( 7 ) , it is possible that kshv - gpcr is causally   involved in tumorigenesis in these diseases . we ( 7 ) had previously suggested that if kshv - gpcr   were shown to be involved in the pathogenesis of primary   effusion lymphoma or ks it would be a target for drug   therapy using a negative antagonist ( or inverse agonist ) . an   inverse agonist is an antagonist that inactivates constitutively active receptors even in the absence of an agonist   ( 35 ) . other investigators have suggested that the protein kinases involved in desensitization of gpcrs may be drug   targets ( 36 ) . in this report , we have shown that coexpression of grk4 , 5 , or 6 as well as direct activation of pkc   inhibit constitutive signaling by kshv - gpcr . moreover ,   inhibition of kshv - gpcr signaling by grks causes inhibition of kshv - gpcr  stimulated cell proliferation ( fig . 5 ) and transformation ( 8) . thus , drug - mediated activation   of grk4 , 5 , or 6 or of pkc might inhibit tumorigenesis . the grks would be the preferred targets as their activation would presumably lead to receptor - specific effects . in conclusion , to our knowledge , this is the first demonstration of grk and pkc inhibition of constitutive signaling by a gpcr associated with human disease . although   the molecular mechanism(s ) of kshv - gpcr desensitization was not proved , these data provide evidence that cell   regulatory pathways of receptor desensitization may be therapeutic targets in diseases involving constitutively active receptors . based on the findings reported here , we suggest   that these protein kinases , along with the kshv - gpcr itself , may be therapeutic targets for pleural effusion lymphomas and ks if kshv - gpcr is shown to be involved in   the pathogenesis of these diseases . effect of coexpression of kshv - gpcr and grks on ip   formation in cos-1 cells . ( a ) comparison of the effects of grk2 , 4 , 5 ,   and 6 . cos-1 cells were cotransfected with plasmids encoding kshv - gpcr ( 3 g / ml ) and one of the grks ( 3 g / ml ) , and ip accumulation   was measured after 90 min . ip accumulation in cells expressing kshv - gpcrs ( control ) is from 410-fold higher than in untransfected or    mock - transfected  ( cells transfected with pcdna3.1(+ ) alone ) cos-1   cells , which was 0.038  0.0046 disintegrations per minute ( dpm ) in ips/ ( dpm in ips + dpm in lipids ) per 90 min . ( b ) effect of expressing various levels of   kshv - gpcr on inhibition of ip accumulation by grk5 . cos-1 cells   were cotransfected with various amounts of plasmid encoding kshv - gpcr ( from 0.1 to 3 g / ml ) in the presence or absence of plasmid encoding grk5 ( 3 g / ml ) . cos-1 cells were cotransfected with   plasmid encoding kshv - gpcr ( 3 g / ml ) or with plasmids encoding   human cxcr2 ( 3 g / ml ) , g16 ( 2 g / ml ) , and a plasmid encoding   grk5 ( 3 g / ml ) . cxcr2 expressing cells were treated with il-8 ( 0.1 m )   chronically ( for 1 or 48 h ) or acutely ( during measurement of ip accumulation ) . there was no difference between cells exposed to il-8 for 1 or 48 h.   human recombinant il-8 was added to a final concentration of 0.25   g / ml . data represent the mean  sem of three experiments . cos-1 cells were transfected with plasmid encoding kshv - gpcr ( 3 g / ml ) in the absence or presence of plasmid encoding grk5   ( 3 g / ml ) and ip accumulation was measured after 90 min . pma was   added 20 min before the experimental incubation at the final concentrations shown . data represent the mean  sd of triplicate determinations   in one of two experiments . additive effects of grk expression and pma on ip formation stimulated by kshv - gpcr . cos-1 cells were transfected with plasmid encoding kshv - gpcr ( 3 g / ml ) in the absence or presence of   plasmids encoding grk2 , 4 , 5 , or 6 ( 3 g / ml ) . pma was added 20 min   before the experimental incubation and ip accumulation was measured   for the subsequent 90 min . effect of expressing grk2 or grk5 on kshv - gpcr stimulated ip accumulation and proliferation in nih 3t3 cells . nih 3t3   cells expressing kshv - gpcrs ( control ) , kshv - gpcrs and grk2   ( + grk2 ) , or kshv - gpcrs and grk5 ( + grk5 ) were used 8 or 9 d   after transfection . for ip experiments , cells were labeled with [ h]myo - inositol for 48 h and ip accumulation was measured during a 90 min incubation . ip accumulation in cells expressing kshv - gpcr alone was   three- to fivefold greater than in mock - transfected cells ( cells transfected   with pcefl ) . data represent the mean  sd of triplicate determinations   in two experiments . for proliferation experiments , cells were harvested   and seeded 5 d after transfection and cells were counted 4 d later .
kaposi 's sarcoma  associated herpesvirus ( kshv)/human herpesvirus 8 , which is consistently   present in tissues of patients with kaposi 's sarcoma and primary effusion lymphomas , contains a   gene that encodes a g protein  coupled receptor ( kshv - gpcr ) . 
 we recently showed that   kshv - gpcr exhibits constitutive signaling via activation of phosphoinositide - specific phospholipase c and stimulates cell proliferation and transformation . in this study , we determined   whether normal cellular mechanisms could inhibit constitutive signaling by kshv - gpcr and   thereby kshv - gpcr  stimulated proliferation . 
 we show that coexpression of gpcr - specific kinases ( grks ) and activation of protein kinase c inhibit constitutive signaling by kshv - gpcr in cos-1 monkey kidney cells and in mouse nih 3t3 cells . 
 moreover , grk-5 but   not grk-2 inhibits kshv - gpcr  stimulated proliferation of rodent fibroblasts . 
 these data   provide evidence that cell regulatory pathways of receptor desensitization may be therapeutic   targets in human diseases involving constitutively active receptors .