dopamine receptors and dopamine signaling have been implicated in various neurological and psychiatric disorders including parkinson 's disease , schizophrenia , and drug abuse [ 13 ] . dopamine receptors are divided into two subfamilies , the gs - coupled d1 and d5 receptors and the gi / o - coupled d2 , d3 , and d4 dopamine receptors that have stimulatory and inhibitory effects on adenylyl cyclase ( ac ) , respectively ( see   for a recent review ) . acute stimulation of d2 dopamine receptors leads to inhibition of ac activity , however , persistent activation of this gi / o - coupled receptor paradoxically results in its enhancement . this phenomenon , called heterologous sensitization of ac , is also known as camp overshoot , supersensitization , or superactivation of ac . d2 dopamine receptor - induced heterologous sensitization of cyclic amp signaling has been demonstrated in several cellular systems as well as in animal models and has also been suggested to occur in humans [ 46 ] . for example , it was observed that repeated administration of the d2 receptor agonist quinpirole enhances ac activity in the caudate putamen , increases creb phosphorylation , and also alters behavior in rodents [ 5 , 6 ] . although this mode of ac regulation has been recognized for over three decades , the molecular signaling mechanism causing heterologous sensitization of ac is only partially understood , attributed to some extent to differences in ac isoform - specific regulation . there are nine differentially regulated membrane - bound ac isoforms in mammalian cells [ 4 , 8 ] . whereas all ac isoforms are stimulated by stimulatory gs , only a subset is inhibited by inhibitory gi , and some ac isoforms are differentially regulated by g [ 4 , 8 ] . here , we studied human adenylyl cyclase type 5 ( ac5 ) that is potently stimulated by gs , inhibited by acute activation of gi , and conditionally activated by g . ac5 is expressed at high levels in the central nervous system and has been identified as a primary effector of d2 dopamine receptors in the striatum [ 9 , 10 ] . the aim of the current study was to investigate the role(s ) of heterotrimeric g proteins in d2 receptor - mediated heterologous sensitization of ac5 . by exploring sensitization in cells devoid of endogenous gs , we were able to examine the ability of gs mutants to support sensitization without interference from endogenous gs . additionally , this gs - deficient cellular model expresses very low levels of ac5 making them a reasonable model for studies of recombinant ac5 . heterologous sensitization of ac5 was readily rescued by wild - type gs and by mutants deficient in palmitoylation   or g interaction . we also assessed the role of g and the signalosome in d2 receptor - induced heterologous sensitization of ac5 by sequestering g subunits with arkct - cd8 [ 15 , 16 ] and coexpressing a dominant - negative mutant of the sar1 gtpase . these experiments revealed that both arkct - cd8 and sar1(h79 g ) attenuated sensitization , suggesting that the components of the signaling complex utilized in heterologous sensitization , presumably ac5 and gs , assemble postsynthesis in the endoplasmic reticulum ( er ) . together with previous findings , the present data support a model in which gs directly interacts with ac5 . in contrast , g appears to have an indirect role in heterologous sensitization of ac5 . the human d2l receptor and ac5 or ac5   were cloned into the dual expression vector pbudce4 ( invitrogen , carlsbad , ca ) creating pbud / hac5 , d2r and pbud/ac5 , d2r . pcdna3/arkct - cd8 [ 15 , 16 ] and pcdna / vsvg - sar1 ( wild type and h79 g )   were used . the c3s mutation was created by site - directed mutagenesis , and the fragment containing the iek+ mutations was amplified by pcr . the resulting constructs , pcdna1/gs - cfp(c3s ) and pcdna1/gs - cfp(iek+ ) were sequenced . all reagents were purchased from sigma - aldrich ( st . louis , mo ) unless otherwise noted . gs - deficient murine embryonic fibroblast cells , gnas cells [ 11 , 12 ] , were a gift from dr . cells were cultured in 50  :  50 mix of f12  :  dmem media supplemented with 5% fbs ( hyclone , logan , ut ) , 1% ant - anti ( invitrogen , carlsbad , ca ) in a humidified incubator at 33c with 5% co2 . approximately 80,000  cells / well were seeded in 24-well plates the day before transient transfection . dna ( 400  ng pbud / hac5 or ac5 , d2r alone or in combination with 10  ng pcdna / gs - cfp , 300  ng pcdna3/arkct - cd8 , or 300  ng pcdna / vsvg - sar1 ) was mixed with opti - mem and 1  l / well lipofectamine 2000 ( invitrogen , carlsbad , ca ) . the medium was replaced with 200  l / well prewarmed opti - mem , and the dna / lipofectamine mixture was added to the cells . after 4  hr , culture medium ( 500  l / well ) was added , and the cells were analyzed after 48  hr . for microscopy , the amount of pcdna / gs - cfp was increased to 100  ng / well . the assays were carried out in assay buffer ( ebss supplemented with 0.2% ascorbic acid , 15  mm hepes , and 2% bcs ( hyclone , logan , ut ) , and 500  m ibmx ) with 100  nm forskolin ( tocris bioscience , ellisville , mo ) as noted for 37c for 15  min . the media was decanted , ice - cold trichloroacetic acid was added , and the lysates were stored at 4c . data were collected from a minimum of three independent experiments carried out in duplicate and were normalized to either basal or vehicle conditions . the graphpad prism 5 software ( graphpad software inc . , lajolla , ca ) was used for data and statistical analyses . the cells were pretreated with 1  m quinpirole or vehicle in assay buffer ( without ibmx ) for 2  hr followed by three washes . camp was measured as described above for acute camp accumulation , with the addition of 1  m spiperone to block the action of any residual quinpirole . a 12 bit photometric coolsnap ( roper scientific ) ccd camera mounted on a te-2000 inverted epifluorescence microscope ( nikon instruments inc . , images were acquired with the metamorph software ( molecular devices , sunnyvale , ca ) and analyzed using image j ( http://rsbweb.nih.gov/ij/ ) . our laboratory has previously shown that mutants of canine ac5 that do not interact with gs are deficient in sensitization [ 23 , 24 ] and that d2-mediated heterologous sensitization of ac5 has an absolute requirement for gs . our present objective was to elucidate the mechanism of gs - dependent heterologous sensitization of human ac5 by utilizing two different gs - cfp   mutants ( figure 1(a ) ) . the c3s substitution eliminates the n - terminal palmitoylation site , which causes gs to mislocalize to the cytosolic fraction . the iek+ mutant contains a series of substitutions , yielding a g-binding deficient gs that also displays a reduction in palmitoylation . the gs - cfp constructs were coexpressed with ac5 and d2 . since both c3s and iek+ are deficient in responses to receptor stimulation [ 13 , 21 ] , we used direct stimulation of ac5 with forskolin throughout this study . basal camp accumulation without any gs was 0.73  0.09  pmol / well , whereas co - expression of gs - cfp increased camp accumulation to 3.12  0.22  pmol / well ( wild - type , wt ) , 4.22  0.06  pmol / well ( c3s ) , and 5.88  0.05  pmol / well ( iek+ ) . forskolin further stimulated camp with values 2.53-fold over basal levels ( figure 1(b ) ) , indicating that wild - type and both gs mutants functionally couple to ac5 . next , expression and subcellular localization of the gs - cfp constructs ( in the presence of ac5 and d2 ) were evaluated by fluorescence microscopy ( figure 1(c ) ) . wild - type gs - cfp showed both plasma membrane and intracellular localization , whereas the c3s and iek+ mutants were predominantly localized intracellularly ( figure 1(c ) ) , consistent with previous reports [ 14 , 21 ] . to assess whether the gs - cfp mutants could rescue heterologous sensitization , cells were pretreated with vehicle or quinpirole followed by camp accumulation . consistent with our previous report , no sensitization of ac5 was observed in the absence of gs ( figure 1(d ) , ctrl ) . in contrast , coexpression of wild - type gs - cfp resulted in robust sensitization of ac5 under both basal and forskolin - stimulated conditions ( figure 1(d ) ) . surprisingly , expression of the gs mutants also significantly rescued heterologous sensitization under basal conditions ( white bars ) and to a lesser degree forskolin - stimulated conditions ( black bars ) . as both mutants are deficient in palmitoylation and membrane localization , neither palmitoylation per se , nor membrane localization of gs appears to be essential for heterologous sensitization of ac5 . although we have established that gs is required for heterologous sensitization , our findings above for the iek+ mutant suggest that direct interactions between gs and g are not critical . this prompted us to further investigate the role of g in d2 receptor - mediated heterologous sensitization of ac5 . the c - terminus of -adrenergic kinase or grk2 ( arkct ) has been used to sequester g subunits and inhibit g-mediated signaling events , including heterologous sensitization [ 15 , 25 , 26 ] . in the absence of arkct - cd8 ( membrane bound arkct ) , sequestering g blocked sensitization of ac5 , under both basal and forskolin - stimulated conditions , revealing the necessity of g for heterologous sensitization of ac5 ( black bars , figure 2(a ) ) . in contrast , arkct - cd8 had no substantial effects on acute d2 receptor activation ; quinpirole produced significant inhibition of camp accumulation in the presence of arkct - cd8 ( 77  10% inhibition ; n = 2 , data not shown ) . in an effort to explore the site of action for g-dependent sensitization this mutant is functional and responds to gs stimulation but is deficient in binding g . the ac5 mutant displayed significant sensitization that was also blocked by arkct - cd8 ( figure 2(b ) ) , suggesting that n - terminal g binding is not intimately involved in heterologous sensitization of ac5 . instead , there are clearly additional , unidentified g interaction sites in ac5 that are necessary for heterologous sensitization . such an assumption is supported by fret and in vitro activation studies of the ac5 deletion mutant   as well as studies of ac2 , which possesses multiple motifs for g interaction and regulation that are located in the c1b and c2b domains of ac2 . other possibilities are that ac5 interacts with endogenous ac isoforms in an ac dimer ( see ) that binds g or that specific g and g subunits or g pairs are involved . however , it is also possible that the g mechanisms involving sensitization of ac may be indirect . because sequestering g subunits alters signalosome assembly , we hypothesized that a specific signaling complex could be required for heterologous sensitization of ac5 . several small gtpases , including sar1 , are involved in signal complex assembly and anterograde protein trafficking . a series of studies using dominant negative mutants of these gtpases shows that gs and g interact with ac2 during trafficking to the plasma membrane [ 30 , 31 ] and that the gs - ac2 interaction is disrupted by sar1(h79 g ) . to study the possibility that interactions between ac5 and its specific signaling partners play a role , we utilized sar1 and sar1(h79 g ) and noted that coexpression with the dominant negative mutant prevented heterologous sensitization of ac5 ( figure 2(c ) ) . in contrast , acute d2 receptor - mediated inhibition of ac5 was not significantly blocked in the presence of sar1(h79 g ) ( data not shown ) . our data are consistent with the findings that sar1(h79 g ) disrupts ac - gs interactions ( as measured by bret or coimmunoprecipitation ) to a larger degree than ac - gi interactions . in contrast , sar1(h79 g ) did not affect the interactions between ac and g , suggesting that the ac interacts with g at an early step in the endoplasmic reticulum ( er ) , but that the interaction with gs occurs after er export . the observation that signaling mechanisms of acute activity and heterologous sensitization are differentially affected further supports the hypothesis that heterologous sensitization and acute stimulation are dependent on separate mechanisms and possibly separate signalosome components . the present data support a complex model of d2 dopamine receptor - induced heterologous sensitization of ac5 where gs appears to directly interact with ac5 . a role for g was confirmed ; however , our observations suggest an indirect role for g that may be involved during the formation of the sensitization signaling complex . a critical role for ac5 in mediating dopamine responses has been previously demonstrated in ac5 deficient mice , which show impaired responses to d2 receptor activation . therefore , these results have implications in brain regions where d2 dopamine receptors and ac5 are coexpressed , such as the striatum , which is implicated in drug addiction , motivation , mood , and voluntary movement . persistent d2 dopamine receptor activation has also been linked to psychiatric disorders ( e.g. , schizophrenia and drug abuse ) and to the adaptive responses associated with drug therapy in parkinson 's disease . enhancing our understanding of the underlying components and mechanisms of heterologous sensitization and regulation of specific ac activity ( in the striatum ) may aid in the development of improved and future therapies for these disorders . for example , recent studies have identified small molecule inhibitors of g-mediated signaling   and ac isoform - specific inhibitors   that may offer novel therapeutic strategies for modulating complex cns behaviors involving dopamine receptor signaling .