BACKGROUND
heterotrimeric guanine nucleotide-binding proteins  are multifaceted signaling modules that relay extracellular signals detected by g protein-coupled receptors  to intracellular effector  <cit> .
at the core of the g protein signal transduction machinery is the gα subunit, a gtpase which acts as a timer to limit the activation signal.
in the classical g protein activation cycle, the gα subunit needs to associate with the gβγ dimer, the gpcr, and effectors separately or simultaneously at different stages of the cycle.
a variety of accessory proteins are now known to modulate the fidelity of the g protein signal.
they include regulators of g protein signaling   <cit> , activators of g protein signaling   <cit> , and adaptor proteins such as tetratricopeptide repeat  <dig>   <cit> .
these additional components allow for rapid inactivation or receptor-independent activation of the gα subunit, as well as signal diversification.
the large number of different types of binding partners for the gα subunit requires optimal utilization of structural domains that are available for protein-protein interactions.
given that gα subunits are typically less than  <dig> kda in size and are attached to the inner leaflet of the plasma membrane, the binding surfaces available for interaction are limited.
nature has partially resolved this constraint by generating different conformations of the gα subunit through the binding and hydrolysis of gtp.
the resolution of the crystal structures of several gα subunits in their gdp- or gtp-bound states  <cit>  and as complexes with the gβγ dimer  <cit>  or other interacting proteins  <cit>  have provided valuable insight into the molecular mechanisms of g protein signal transduction.
structurally, the gα subunit can be broadly divided into the gtp hydrolase  and helical domains  with the former harboring the gtp-binding pocket  <cit> .
several regions  spreading across the gtpase and helical domains exhibit profound conformational changes when the gα subunit shifts between the gdp- and gtp-bound states  <cit> .
changes in the switch regions provide the molecular basis of g protein activation and effector regulation.
in the gtp-bound active state, the gα subunit releases the gβγ dimer and thus allows effectors to bind to the newly exposed surfaces such as the switch ii region  <cit> .
this simplistic view, however, cannot accommodate the increasing numbers of gα-interacting proteins.
the activated gα subunit is a preferred partner for multiple effectors, adaptors, and rgs proteins.
a central question is whether an activated gα subunit can concurrently regulate multiple signaling pathways by simultaneously binding to different partners in much the same way as an inactive gα subunit forms a complex with the gβγ dimer and the receptor.
among the different subfamilies of gα subunits, members of the gαq subfamily have the capacity to activate phospholipase cβ   <cit>  as well as interact with the guanine nucleotide exchange factor p63rhogef  <cit> , g protein-coupled receptor kinase grk <dig>  <cit> , adaptor proteins such as tpr <dig>  <cit> , and several rgs proteins  <cit> .
these molecules bind to overlapping as well as distinct regions on gαq .
it is often assumed that the primary signal generated by gq-coupled receptors is the formation of inositol trisphosphates  by plcβ, and that the regulation of downstream kinases and transcription factors are consequential to the production of ip <dig> and the subsequent ca2+ mobilization.
however, recent studies suggest that gα subunits can concurrently regulate multiple signaling pathways.
the ability of gα <dig>  a member of the gαq subfamily, to interact with the adaptor protein tpr <dig>  <cit>  has raised some interesting scenarios.
gα <dig> is primarily expressed in hematopoietic cells and it can regulate multiple signaling pathways  <cit> .
interestingly, tpr <dig> can directly interact with ras especially when the latter is activated  <cit>  and it appears to link gα <dig> to ras activation  <cit> .
ras is a small gtpase which acts as a molecular switch for linking various cell surface receptors to intracellular signaling pathways, resulting in cell proliferation and differentiation  <cit> .
we have recently demonstrated that constitutively active gα <dig>  induces the phosphorylations of transcription factors, such as signal transducer and activator of transcription  <dig>  and nuclear factor κb , through plcβ and ras/raf-1/mek/erk signaling cascades in human embryonic kidney  <dig>  cells  <cit> .
however, it is not known if the binding of tpr <dig> to gα <dig> affects plcβ signaling and whether tpr <dig> and plcβ utilize the same docking site on gα <dig>  although gα <dig> has been shown to interact with the c-terminus of tpr <dig>  <cit> , the structural requirement for gα <dig> to interact with tpr <dig> has yet to be defined.
in the present study, we examined the structural domain of gα <dig> for interacting with tpr <dig> and assessed whether the same domain is responsible for regulating plcβ.
methods
materials
the human cdnas of gα <dig>  gα16ql, gαz and gαzql were obtained from missouri s&t cdna resource center.
c <dig> and c <dig> cdnas were previously constructed and characterized  <cit> .
cell culture reagents, including lipofectamine™ and plus™ reagents, and accuprime™ pfx supermix were purchased from invitrogen .
anti-gα <dig>  and anti-gαz  were obtained from gramsch laboratories .
anti-gα <dig>  was purchased from torrey pines biolabs .
anti-gαz  and plcβ antibodies were from santa cruz biotechnology .
unbound and affinity gel-conjugated anti-flag antibody were from sigma-aldrich .
other antibodies were purchased from cell signaling technology .
protein g-agarose and protein cross-linking agent dithiobis were from pierce biotechnology .
ecl kit was from ge healthcare bio-sciences .
ras activation kit was a product of millipore .
construction of chimeras
gα chimeras were constructed from the cdnas encoding the human gα <dig> and gαz by using pcr techniques.
the n-terminal  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> residues of gα <dig> were substituted by the corresponding regions of gαz to generate n <dig>  n <dig>  n <dig>  n <dig>  n <dig>  n <dig>  n <dig> and n <dig> chimeras, respectively.
primer pairs were designed to cover the overlapping regions in forward and reversed directions.
for each construct, the 5' fragment was generated with the reversed and t <dig> primers, whereas the 3' fragment was made with the forward and sp <dig> primer.
the two half-products were then annealed together to generate a full-length fragment by another round of pcr using t <dig> and sp <dig> primers.
mirror-images of these constructs were generated analogously and were named c <dig>  c <dig>  c <dig>  c <dig> and c <dig> chimeras.
primers for chimera construction are listed in table  <dig>  pcr was carried out using accuprime™ pfx supermix .
the n200-c <dig> and n188-c <dig> chimeras were constructed using c <dig> as the initial template for the 3' half-products and those primers designed for n <dig> and n <dig>  respectively.
likewise, zβ <dig> and zβ2β <dig> were constructed using the c <dig> and c <dig> primers with n <dig> as the initial templates.
all chimeras were checked by restriction mapping and then subcloned into pcdna <dig> at hind iii and xho i sites.
the constructs were fully sequenced by dideoxynucleotide sequencing to confirm the identities.
bold and italic nucleotides denote the gα16-derived sequences.
cell culture and co-immunoprecipitation experiments
hek  <dig> cells were obtained from the american type culture collection .
they were maintained in eagle's minimum essential medium at 5% co <dig>  37°c with 10% fetal bovine serum ,  <dig> units/ml penicillin and  <dig> μg/ml streptomycin.
hek  <dig> cells were grown to 80% confluency in 100-mm tissue culture plates and then co-transfected with  <dig> ng gα and  <dig> ng flag-tpr <dig> cdnas using  <dig> μl plus™ and lipofectamine™ reagents in opti-mem™.
fbs was replenished  <dig> h after transfection.
cross-linking was performed one day after transfection.
transfected cells were washed with pbs twice and then treated with  <dig>  mm dithiobis in pbs for  <dig> min at room temperature.
cells were then washed again with pbs and maintained in quenching solution  for  <dig> min.
subsequently, cells were lysed in ice-cold ripa buffer .
cell lysates were gently rocked with an anti-gα <dig>  anti-gαz or anti-plcβ antiserum at 4°c overnight, and then incubated in  <dig> μl protein g-agarose  at 4°c for  <dig> h. alternatively, the cell lysates were incubated in  <dig> μl anti-flag affinity agarose gel  at 4°c overnight.
immunoprecipitates were washed with ice-cold ripa buffer  for four times, resuspended in  <dig> μl ripa buffer and  <dig> μl  <dig> × sample buffer and then boiled for  <dig> min.
gα <dig>  gαz, plcβ and flag-tpr <dig> proteins in the immunoprecipitates were analyzed by western blots.
ras activation assay
hek  <dig> cells were co-transfected with  <dig> ng gα,  <dig> ng flag-tpr <dig> and  <dig> ng ras cdnas.
after  <dig> day, transfectants were serum-deprived for  <dig> h. cells were then washed twice with ice-cold pbs and lysed with the  <dig> × mg2+ lysis buffer .
cell lysates were pre-cleaned with protein g-agarose and activated ras was immunoprecipitated with  <dig> μl raf- <dig> ras-binding domain-conjugated agarose  for  <dig> min and followed by three washes of  <dig> μl ice-cold mlb.
immunoprecipitates was finally reconstituted in  <dig> μl mlb and  <dig> μl  <dig> × sample buffer and resolved in sds-page for detecting ras using specific antibody.
assays for phosphorylated erk, ikk and stat3
hek  <dig> cells were seeded on a 6-well plate at  <dig>  ×  <dig> cells/well  <dig> day prior to transfection.
transfection was performed with  <dig> ng gα and  <dig> ng flag-tpr <dig> cdnas using  <dig> μl plus™ and lipofectamine™ reagents in opti-mem™.
the transfectants were serum-deprived overnight one day after transfection.
cells were treated with pertussis toxin  and n6-cyclohexyladenosine  where appropriate, lysed, and then assayed for phosphorylation statuses of erk, stat <dig>  and ikk as described previously  <cit> .
inositol trisphosphate accumulation assay
hek  <dig> cells were seeded on a 12-well plate at  <dig> ×  <dig> cells/well one day prior to transfection.
cells were transfected with  <dig> ng gα with or without  <dig> ng type  <dig> adenosine receptor  cdnas using  <dig> μl lipofectamine™  <dig> reagent in opti-mem™ containing 5% fbs.
transfectants were labeled with  <dig>  μci/ml myo-inositol and subsequently assayed for cha-induced ip <dig> formation as described previously  <cit> .
nfκb-driven and stat3-driven luciferase assays
for stat3-driven luciferase assay  <cit> , hek  <dig> cells were seeded on a 96-well microplate at  <dig>  cells/well one day before transfection.
cells were transfected with  <dig> ng gα and  <dig> ng pstat3-luc luciferase reporter using  <dig>  μl plus™ and lipofectamine™ reagents in opti-mem™ and fbs was replenished after  <dig> h. when a1r-induced signals were analyzed,  <dig> ng receptor cdna was included in the transfection.
for nfκb-driven luciferase assay, hek  <dig> cells stably transfected with pnfκb-luc luciferase reporter were seeded on a 96-well microplate at  <dig>  cells/well.
the setup and transfection were as described previously  <cit> .
one day after transfection, transfectants were serum-deprived for  <dig> h and ptx  was added where necessary.
cells were challenged with or without  <dig> μm cha for  <dig> h before measuring the luciferase activity as reported previously  <cit> .
western blotting analysis
protein samples were resolved on 12% sds-page and transferred to nitrocellulose membrane .
resolved proteins were detected by their specific primary antibodies and horseradish peroxidase-conjugated secondary antisera.
the immunoblots were visualized by chemiluminescence with the ecl kit from ge healthcare bio-sciences, and the images detected in x-ray films were quantified by densitometric scanning using the eagle eye ii gel documentation system .
molecular modeling of gα subunits
gαq in the complex with p63rhogef and rhoa   <cit>  was employed for the illustration of functional domains of gαq, and for creating a molecular model of gα <dig> by homologous modeling using swiss-model  <cit> , which allowed manual adjustments to the alignment of the sequences of gα <dig> with gαq in order to accommodate the extraordinarily long α4/β <dig> loop of gα <dig>  molecular models of gαz with or without the mutations at β2/β <dig> loop  were generated using 3d-jigsaw  <cit>  based on the crystal structure of gαi <dig> bound to alf4- and gdp   <cit>  as selected by the default automatic mode.
3d-jigsaw-generated models showed greater dynamics in the loop structures and allowed for the exploration of potential conformational variations caused by the mutations.
visualization of various structures was accomplished using ucsf chimera  <cit> .
RESULTS
extreme termini of gα <dig> are not involved in tpr <dig> interaction
unlike the receptor-interacting domain which is composed of five distinct structures  <cit> , no discrete localization for effector interaction is generally applicable to the gα subunits, probably because the different signaling pathways entail a diverse spectrum of effector molecules.
the gα <dig> regions responsible for effector interaction have not been mapped, but there is evidence to suggest that the switch iii and the α <dig> helix may participate in the binding of p63rhogef  <cit>  and that the plcβ-interacting domain on gαq mainly encompasses the α2-β4-α3-β <dig> regions  <cit> .
the involvement of β <dig> strand, α <dig> and α <dig> helices in the gαq/plcβ complex formation has been revealed clearly in the very recently resolved crystal structure  <cit>  which also confirmed that plcβ can serve as a gap for gαq  <cit> .
in order to identify the structural domains on gα <dig> that interact with tpr <dig>  gα <dig> sequences were progressively replaced by those of gαz because the latter does not recognize tpr <dig> .
chimeras composed of gα <dig> and gαz residues were preferred because they are structurally viable  <cit> .
a series of chimeras was made by swapping discrete regions between gα <dig> and gαz.
construction of the chimeras was guided by the predicted tertiary structure of the gα subunits as well as by our previous experience in determining the receptor and effector interacting domains of gα <dig> and gαz  <cit> .
for chimeras with substitutions at either the n- or c-terminus, they were named with a single letter  followed by the numbers of amino acids of gαz that substituted the corresponding regions of gα <dig>  the two chimeras containing either β <dig> or β2-β <dig> strands of gαz were named with the a letter "z" followed by "β2" or "β2β3", respectively, in order to distinguish them from the descriptions of specific β strand structures.
we first examined two chimeras of gα <dig> containing either  <dig> or  <dig> amino acids of gαz at the c-terminus; representing changes in the α <dig> helix alone and α4/β <dig> loop plus the α <dig> helix, respectively.
these two chimeras, named c <dig> and c <dig> respectively , have been previously constructed and characterized .
both chimeras exhibit enhanced coupling to gi-coupled receptors and possess the ability to stimulate plcβ.
hek  <dig> cells were co-transfected with flag-tpr <dig> in combination with pcdna <dig>  gα <dig>  c <dig> or c <dig>  as illustrated in figure 2c , gα <dig>  c <dig> and c <dig> were co-immunoprecipitated with the anti-flag affinity gel with similar levels of flag-tpr <dig>  moreover, flag-tpr <dig> was coimmunoprecipitated along with gα <dig>  c <dig> or c <dig> .
gα <dig>  c <dig>  c <dig> and flag-tpr <dig> were expressed at detectable and comparable levels in the transfectants .
since both chimeras retained the ability to interact with flag-tpr <dig>  it implies that the c-terminal β <dig> and α <dig> regions of gα <dig> are not required for the interaction with tpr <dig>
the n-terminus of gα subunits participates in membrane attachment, gβγ binding as well as receptor recognition.
another previously constructed chimera, n <dig>  <cit> , was employed to test the possible involvement of the n-terminus of gα <dig> in tpr1-binding.
the αn helix  of n <dig> is composed of gαz sequence .
as shown in figure 2d , both gα <dig> and n <dig> co-immunoprecipitated with flag-tpr <dig> and similar levels of flag-immunoreactivity were observed.
similarly, flag-tpr <dig> co-immunoprecipitated with gα <dig> and n <dig> and the levels of these gα subunits in the immunoprecipitates were essentially the same .
like gα16ql, constitutively active n30ql was more readily associated with tpr <dig>  and this effect was not due to variations in expression levels .
given that n <dig> still possessed the ability to interact with flag-tpr <dig>  our results suggest that the region which is critical for direct or indirect binding of tpr <dig> may lie between the β <dig> strand and α <dig> helix of gα <dig>
the β <dig> region of gα <dig> interacts with tpr1
to further examine the structural domain of gα <dig> for tpr <dig> interaction, additional chimeras were constructed by replacing different regions of gα <dig> with those of gαz.
four different chimeras named n <dig>  n <dig>  n <dig> and n <dig> were thus constructed  by replacing a gαz backbone with gα <dig> sequences starting from the α <dig>  α <dig>  β <dig>  and α <dig> regions, respectively.
however, none of the four gα <dig> chimeras could be pulled down by anti-flag affinity gel .
similarly, flag-tpr <dig> could not be co-immunoprecipitated by anti-gα <dig> antiserum in chimera-expressing cells  despite detectable expression levels of flag-tpr <dig> and the chimeras in the cell lysates .
in control experiments, both gα <dig> and gα16ql were co-immunoprecipitated with flag-tpr <dig> .
these findings demonstrate that the tpr1-interacting domain must reside between residues  <dig> and  <dig> that represent the regions from β <dig> to β <dig> of gα <dig>
two more chimeras were constructed to map the tpr1-interacting domain within the first 30- <dig> residues.
chimeras n <dig> and n <dig> were made by replacing the n-terminus of gα <dig> with gαz sequences up to and including the αa and αd regions, respectively .
both n <dig> and n <dig> were found to co-immunoprecipitate with flag-tpr <dig> .
association of these two chimeras with flag-tpr <dig> was confirmed by reverse co-immunoprecipitation using the anti-gα <dig> antiserum .
these results narrowed down the tpr1-interacting domain of gα <dig> to be among the αe to β <dig> regions.
before pinpointing the precise location of the tpr1-interacting site on gα <dig>  we first confirmed the preceding observations with additional chimeras that represent the mirror images of several tested chimeras.
chimeras c <dig>  c <dig>  and c <dig> were essentially the mirror images of n <dig>  n <dig>  and n <dig>  respectively , and they should exhibit phenotypes opposite to those of their counterparts.
indeed, c <dig> and c <dig> could not associate with flag-tpr <dig> even though they were efficiently expressed .
conversely, chimera c <dig> should be able to interact with flag-tpr <dig> because its mirror image  failed to associate with flag-tpr1; co-immunoprecipitation of c <dig> and flag-tpr <dig> confirmed their association .
these results again indicate that the tpr1-interacting domain of gα <dig> lies within the αe-αf-β2-β <dig> regions which are common in n <dig>  n <dig>  and c <dig> but missing in their corresponding mirror images .
to tease out the tpr1-interacting domain, two new constructs were made by dissecting the αe-αf-β2-β <dig> regions into two halves.
c <dig> contained the n-terminal half of gα <dig> up to and including the αe-αf regions, while n <dig> represented its mirror image and contained gα16-specific sequence from the β2-β <dig> regions onward .
because these two mirror images contained completely different regions of gα <dig> and gαz, two distinct anti-gα <dig> antisera, targeting either the extreme n-terminus  or c-terminus , were required in the co-immunoprecipitation assay.
although both mutants were expressed to comparable levels , only n <dig>  was co-immunoprecipitated with flag-tpr <dig> .
for the reverse co-immunoprecipations, n <dig> and n188ql, but not c <dig> and its mutant, were able to interact with tpr <dig> .
since these findings suggest that only β2-β <dig> within the αe-αf-β2-β <dig> region is responsible for tpr <dig> association, two more chimeras  were created to split this region into two halves ; c <dig> possessed gα16-specific β <dig> region whereas n <dig> harbored the gα16-specific β <dig> region.
co-immunoprecipitation experiments with flag-tpr <dig> illustrated that n <dig> and its constitutively active mutant n200ql interacted with flag-tpr <dig> while c <dig> and c174ql were ineffective .
reverse co-immunoprecipitations with anti-gα <dig> antisera confirmed the ability of tpr <dig> to associate with n <dig> and n200ql.
in order to validate the importance of the β <dig> region of gα <dig> for tpr <dig> interaction, n188-c <dig> and n200-c <dig> were constructed as shown in figure 6a.
n188-c <dig> is primarily a gαz backbone with the β <dig> and β <dig> regions from gα <dig>  while n200-c <dig> is essentially gαz with the β <dig> region made up of the gα <dig> sequence.
co-immunoprecipitation experiments using anti-flag or anti-gαz antisera showed that both chimeras and their respective constitutively active mutants interacted with flag-tpr <dig> .
association of n200-c <dig> with flag-tpr <dig> suggests that the β <dig> region of gα <dig> alone is sufficient to confer upon gαz the ability to interact with tpr <dig>  on the other hand, replacement of the β <dig> region of gα <dig> with the cognate sequence of gαz is expected to disrupt gα16/tpr <dig> interaction.
two additional chimeras, named zβ <dig> and zβ2β <dig>  with either the β <dig> or β2-β <dig> regions of gαz inserted into a gα <dig> backbone  were constructed to test this hypothesis.
zβ2β <dig> and zβ2β3ql failed to co-immunoprecipitate with tpr <dig> , thus demonstrating the importance of the β <dig> and β <dig> regions of gα <dig> for interaction with tpr <dig>  however, very weak but detectable associations of zβ <dig> and zβ3ql with flag-tpr <dig> were observed in co-immunoprecipitation assays .
these results confirm the crucial role of the β <dig> region for the gα16/tpr <dig> interaction, and further suggest that the β <dig> region may facilitate the actions of the β <dig> strand.
activation of ras via the association of tpr <dig> with gα <dig> and its chimeras
association of tpr <dig> with ras may provide a more direct link for gα <dig> to activate the erk cascade instead of going through the plcβ/pkc pathway.
if the β <dig> region of gα <dig> is essential for functional interaction with tpr <dig> and ras, chimeras containing this region should facilitate the activation of ras while those lacking this region ought to be inactive.
as predicted, transfectants expressing n200ql  exhibited elevated ras activity whereas c174ql  did not activate ras .
to extend this study, other tpr1-interacting chimeras were evaluated .
no significant ras activation was detected when wild-type n <dig>  n <dig>  n <dig> and c <dig> were overexpressed.
upon the introduction of the ql mutants of these chimeras, all chimeras induced detectable ras activation as compared to their corresponding wild-type counterparts .
likewise, n188-c <dig> and n200-c <dig> should be capable of activating ras because they possess the β <dig> region of gα <dig>  indeed, the constitutive active mutants of these chimeras activated ras .
conversely, zβ2β <dig> and zβ <dig>  did not stimulate the ras activity .
these results indicate that chimeras containing the gα16-specific β <dig> region possess the ability to activate ras, and such activity is dependent on the gtp-bound conformation of the chimeras.
gα16ql-induced ras activation is independent of plcβ signaling
the plcβ-interacting domain of gαq was initially mapped to residues 217- <dig>  <cit> , corresponding to the α2-β4-α3-β <dig> regions of gα <dig>  recent resolution of the crystal structure of gαq-plcβ has refined the plcβ-interaction surface on gαq to encompass mainly β <dig> strand, α <dig> and α <dig> helices  <cit> .
since the putative plcβ-interacting domain of gα <dig> is in proximity of the tpr1-interacting β <dig> region, we asked if activation of ras and plcβ can occur independently.
the chimeras were transiently expressed in the absence or presence of the gi-coupled a1r and then assayed for ip formation with or without  <dig> μm cha.
predictably, cha-induced ip formation was observed with transfectants co-expressing gα <dig>  n <dig>  c <dig>  or c <dig> .
chimeras with parts of the plcβ-interacting regions of gα <dig> replaced by cognate gαz sequences should exhibit impaired ability to regulate plcβ.
such chimeras include n <dig>  n <dig>  n <dig>  c <dig>  c <dig>  c <dig>  c <dig>  c <dig>  n188-c <dig>  and n200-c <dig>  indeed, all ten chimeras failed to stimulate plcβ in response to cha , although cha was capable of inhibiting camp formation in the transfectants .
the putative plcβ-interacting region of gα <dig> is intact in n <dig>  n <dig>  n <dig>  n <dig>  n <dig>  zβ2β <dig>  and zβ <dig>  and hence these chimeras are expected to support a1r-mediated ip formation.
however, only transfectants harboring n <dig>  zβ2β <dig> or zβ <dig> responded to cha with a significant increase in ip formation .
the lack of response to cha challenge may be attributed to impairment in receptor/g protein recognition.
to exclude such a possibility, we tested the ability of the constitutively active mutants of the chimeras to stimulate plcβ.
those mutant chimeras with the plcβ-interacting region disrupted or replaced by gαz residues did not exhibit any stimulation of plcβ, while gα16ql and the previously characterized mutants such as c <dig> efficiently induced ip formation in the transfectants .
among the mutant chimeras with the α2-β4-α3-β <dig> region intact, only n188ql, n200ql, zβ2β3ql and zβ3ql constitutively stimulated the plcβ activity .
the constitutive activity of n200ql suggests that the inability of n <dig> to mediate a1r-induced stimulation of plcβ may be attributed to defective recognition of receptor.
with the exception of n <dig>  n <dig>  and n <dig>  the plcβ-stimulating abilities of the chimeras were in general agreement with the predicted presence of the putative plcβ-interacting region.
hek  <dig> cells were co-transfected with a1r and pcdna <dig> or the indicated gα subunit.
transfectants were labeled with inositol  in dmem containing 5% fbs overnight.
ip <dig> formations were examined in the absence  or presence of  <dig> μm cha for  <dig> min.
fold stimulations were calculated as the ratios of cha-induced to basal ip <dig> accumulations or ql-induced to wild-type ip <dig> accumulations.
data represent the mean ± s.d.
of triplicate determinations of a single representative experiment, n = 3; significant responses are shown in bold and italic.
constructs containing an intact putative plcβ binding domain  of gα <dig> are marked with a "yes".
* cha-stimulated ip <dig> production is significantly greater than basal ; paired t-test, p ≤  <dig> .
# ql-stimulated ip <dig> production is significantly greater than the corresponding wild-type activity; paired t-test, p ≤  <dig> .
a ql-induced fold stimulation of 16z <dig> was extrapolated from  <cit> .
to confirm that the plcβ-interacting regions of gα16-interaction are distinct from that of tpr <dig>  two series of gα <dig> chimeras were tested.
the first series contained chimeras harboring the putative plcβ-interacting domains, including n <dig>  n <dig>  and n <dig>  hek  <dig> cells were cotransfected with plcβ <dig> and gα <dig> or a chimera and then subjected to co-immuniprecipitation using antisera against either gα <dig> or plcβ <dig>  interaction between plcβ <dig> and gα <dig> was clearly evident, with gα16ql generating a stronger interaction with plcβ <dig> .
chimeras n <dig>  n <dig>  and n <dig> were all capable of being co-immunoprecipitated with plcβ <dig>  another set of chimeras including c <dig>  c <dig>  and c <dig>  was similarly tested .
due to the replacement of the putative plcβ-interacting regions with sequences of gαz, these chimeras were expected not to interact with plcβ <dig>  indeed, even with successful expression of the chimeras and plcβ <dig>  no plcβ <dig> interaction was detectable for these chimeras .
these results support the notion that the β <dig> region of gα <dig> is dispensable for the interaction with plcβ .
since several chimeras can apparently activate ras through tpr <dig> despite their inability to stimulate plcβ, they represent useful tools in delineating complex signaling networks such as those for the regulation of stat <dig> and nfκb.
if these chimeras can induce the phosphorylation and activation of stat <dig> and nfκb, then it would imply that plcβ activity is not essential.
conversely, a lack of activity on stat <dig> and nfκb by these chimeras would indicate that plcβ action is required.
transfectants co-expressing the chimeras and a1r were stimulated with cha, and the phosphorylation of stat <dig> and inhibitor of κb kinase  monitored by western blot analysis using phosphoprotein-specific antibodies.
as shown in table  <dig>  chimeras with dual tpr1/ras and plcβ activating capabilities effectively mediated cha-induced stat <dig>  and ikk phosphorylations, with magnitudes similar to those of gα <dig>  these chimeras included n <dig>  n <dig>  c <dig>  c <dig>  zβ2β <dig>  and zβ <dig>  n <dig> failed to mediate the cha-induced stat <dig> phosphorylation but supported the ikk phosphorylation .
tpr1-interacting chimeras which lack plcβ activity  were unable to support cha-induced phosphorylation of stat <dig> and ikk .
because erk is required for gα16-mediated stat <dig> and nfκb activation  <cit> , we further tested the ability of the chimeras to mediate erk phosphorylation.
the profile of erk phosphorylation mediated by the chimeras closely resembled those for stat <dig> and ikk .
chimeras lacking both plcβ and tpr1/ras activities, including n <dig>  n <dig>  n <dig>  n <dig>  c <dig>  c <dig>  c <dig>  and c <dig>  did not support any of the cha-induced responses, whereas n <dig>  n <dig>  n <dig>  c <dig>  c <dig>  zβ2β <dig>  and zβ <dig> mediated cha-induced erk phosphorylation .
hek  <dig> cells were co-transfected with adenosine a1r and pcdna <dig> or the indicated gα subunit.
transfectants were serum starved overnight in the presence of  <dig> ng/ml of ptx and then challenged with  <dig> μm cha for  <dig> min.
cell lysates were then resolved and immunoblotted against various specific antibodies.
results are expressed as fold stimulation over the basal , n = 3; significant responses are shown in bold and italic.
constructs shown to co-immunoprecipitate with flag-tpr <dig> or stimulate plcβ activity  are marked with a "yes".
* cha-stimulated phosphorylation of the target protein is significantly greater than the basal; paired t-test, p ≤  <dig> .
a significant plcβ activation observed with the ql mutant only .
b very weak association with tpr <dig> was detected in co-immunoprecipitation assays .
finally, we employed luciferase reporter gene assays to demonstrate transcriptional regulation of stat <dig> and nfκb by those chimeras that possess dual tpr1/ras and plcβ activating capabilities.
in agreement with our previous studies  <cit> , cha induced stat3- and nfκb-driven luciferase activities in transfectants co-expressing gα <dig> but not gαz .
n <dig>  n <dig>  n <dig>  c <dig>  c <dig>  and zβ <dig> chimeras all supported cha-induced stat3- and nfκb-driven luciferase activities, whereas no transcriptional activation was observed in transfectants co-expressing chimeras that failed to mediate stat <dig>  ikk, and erk phosphorylations .
collectively, these studies demonstrate that dual tpr1/ras and plcβ activating capabilities of gα <dig> may be essential for its regulation of complex signaling networks such as those for the activation of stat <dig> and nfκb.
hek  <dig> cells were co-transfected with adenosine a1r, pstat3-luc/pnfκb-luc, and pcdna <dig> or the indicated gα subunit.
transfectants were serum starved for  <dig> h in the presence of  <dig> ng/ml of ptx and then challenged with  <dig> μm cha overnight.
cell lysates were analyzed for luciferase activity.
data represent the mean ± s.d.
of triplicate determinations, n = 3; significant stimulations are shown in bold and italic.
* cha-stimulated protein phosphorylation is significantly greater than the basal; paired t-test, p ≤  <dig> .
discussion
protein-protein interactions are central to the functions of gα subunits and each gα subunit has to coordinate such interactions in a timely manner according to its guanine nucleotide binding state.
once activated, the gtp-bound gα subunit has a limited time-span to regulate downstream effectors before its intrinsic gtpase activity turns it back to the inactive gdp-bound state, a process which can occur rapidly in the presence of rgs proteins.
hence, simultaneous regulation of multiple signaling pathways by activated gα <dig> is desirable but this entails the deployment of different binding surfaces.
although ras can be activated indirectly by plcβ signaling, our results provide a structural basis for gα <dig> to stimulate ras through interaction with tpr <dig> instead of plcβ.
moreover, functional analyses of gα16/z chimeras reveal that dual stimulation of tpr1/ras and plcβ may be essential for gα <dig> to activate downstream transcription factors such as stat <dig> and nfκb.
the extensive array of gα16/z chimeras has enabled us to pinpoint the tpr1-interacting domain to the β <dig> region  of gα <dig>  the β2-β <dig> region lies between the switch i  and ii  regions  <cit>  and is accessible for protein-protein interaction .
since activation of the gα subunit alters the conformation of several switch regions drastically, proteins like plcβ that bind to the switch regions will interact with the gtp-bound active state much effectively than the gdp-bound basal state of the gα subunit .
however, the differences of the interactions between tpr <dig> and gα16wt or gα16ql are comparably smaller, and tpr <dig> can obviously bind to gα16wt.
such observation implies that tpr <dig> may bind to gα <dig> at regions with relatively less drastic conformational changes.
our results suggested that the β <dig> strand of gα <dig> alone appears to be sufficient for the interaction with tpr <dig>  the mere incorporation of the β <dig> region of gα <dig> confers upon gαz the ability to bind tpr <dig> .
n200-c <dig> has the amino acid sequence 201ktnlrivdvg from gα <dig> inserted into a gαz backbone, but only the first six residues are different from the corresponding gαz sequence .
based on the crystal structure of gαi <dig>  <cit> , molecular modeling of gαz reveals that glu <dig> is located at the "hook-shaped" β2-β <dig> turn , which is often composed of two oppositely charged residues flanked by two interacting hydrophobic residues except gα <dig>  with a threonine following the lys <dig>  the predicted β2-β <dig> turn of n200-c <dig> showed a more widened conformation , which presumably forms a characteristic microdomain on gα <dig> for tpr <dig> interaction.
the fact that the chimera zβ <dig> exhibited residual binding to tpr <dig>  suggested that other regions like the β <dig> strand may also participate in the binding of tpr <dig> because its removal in the zβ2β <dig> chimera can further suppress tpr <dig> binding as compared to zβ <dig> .
since several chimeras failed to generate a response in all of the functional assays, these chimeras might not be able to adopt the active conformation properly and it remains possible that additional residues other than the β <dig> region may bind to tpr <dig>
although the chimeras used in the present study were not tailored for the mapping of the plcβ-interacting domain of gα <dig>  they nonetheless proved useful in locating the overall site for the binding of plcβ.
as in the case of gαq  <cit> , the regions comprising of β <dig>  β <dig>  α <dig>  and α <dig> are likely to form the plcβ-interacting surface of gα <dig>  because chimeras with one or more of these regions disrupted all failed to stimulate ip formation .
a more interesting observation is that chimeras including n <dig>  n <dig>  n <dig>  and n <dig> could not activate plcβ at all, and only the constitutively active form of n <dig> could stimulate plcβ weakly, whereas the chimera n <dig> recapitulates the plcβ-activating capability.
n <dig> and n <dig> contained all the putative plcβ-interacting domains and yet could not activate plcβ, albeit both interacted with tpr <dig> and activated ras.
one plausible explanation is that both chimeras have a hybrid helical domain  which may affect its structural and functional integrity.
indeed, all gα16/z chimeras that can stimulate plcβ activity  contain a contiguous helical core  in addition to the putative plcβ-interacting domains.
an early study of gαs suggested that its helical domain could function as an internal gap for the gtpase domain  <cit> , and the intra-molecular interaction between the gtpase and helical domains was proven to be essential for guanine nucleotide binding and receptor-mediated activation  <cit> .
furthermore, the αc-αd loop of gα subunits has been recognized as switch iv which shows significant conformational changes in different guanine nucleotide-binding states and could reduce the nucleotide exchange rate when mutated  <cit> .
it is therefore possible that a hybrid helical domain may impede the activation of plcβ by the gα16/z chimeras.
the sudden regain of plcβ-activating property of n <dig> which contains a helical domain completely derived from gαz also implies that structural integrity of the helical domain is critical for plcβ activation.
as revealed in the very recent crystal structure of gαq-plcβ <dig>  <cit> , the β <dig> and β <dig> strands of gαq interact extensively with the c <dig> domain of plcβ <dig>  and these two strands also heavily contribute to the overall scaffold of the gtpase domain.
replacement of one or both of them, as in the n <dig> and n <dig> chimeras, might disrupt plcβ interaction severely.
nonetheless, n <dig> can successfully form a heterotrimer with flag-tagged gβγ dimer in co-immunoprecipitation experiment , excluding the possibility of improper folding of the chimera.
a molecular model of gα <dig> is constructed by homologous modeling based on the crystal structure of gαq to visualize the potential interacting surfaces for plcβ and tpr <dig>  except for the unusually long α4-β <dig> loop and differences in the n-terminal helix , gα <dig> basically fit very well to the structure of gαq.
assuming that gα <dig> utilizes domains similar to those of gαq for the binding of plcβ  <cit> , it is entirely feasible for gα <dig> to simultaneously regulate plcβ and tpr <dig>  as shown in the right panel of figure 9a, the critical plcβ-interacting residues are clustered mostly on the left hand side of the gtpase domain of gα <dig> , while the putative tpr1-interacting β <dig> region is located in the lower quadrant of the right hand side without severe overlap with the plcβ-interacting region.
preliminary co-immunoprecipitation studies indeed suggest the existence of gα16/plcβ and plcβ/tpr <dig> complexes .
further studies will be required to confirm if a gα16/plcβ/tpr <dig> complex truly exists.
tpr motif-containing adaptors such as rapsyn are known to cluster signaling molecules for the efficient propagation of signals  <cit> .
it is conceivable that tpr <dig> may serve a similar function in g protein pathways.
its lack of association with gα <dig>  gαt <dig>  <cit>  and gαz  suggests that tpr <dig> can selectively link g protein signals to ras-dependent pathways.
for those that can interact with tpr <dig>  it remains to be determined if binding to tpr <dig> confers upon them the same repertoire of signaling capabilities.
the distinct locations for plcβ- and tpr1-interacting regions infer that gα <dig> can regulate them independently.
indeed, we have demonstrated that some chimeras  can bind tpr <dig>  and induce ras activation  even though they lack the ability to stimulate plcβ .
this raises an interesting possibility that, depending on the composition of the signaling modules within a cell, activation of gα <dig> may differentially regulate tpr1/ras and plcβ signaling pathways.
both ras and plcβ activities are apparently required for the regulation of stat <dig> and nfκb  <cit>  , but it is not clear if the two components are arranged in parallel or in series.
given that the activation of stat <dig> and nfκb could only be detected with chimeras possessing the ability to activate both ras and plcβ, it would seem that the two pathways are independently required for the regulation of stat <dig> and nfκb.
the need for multiple input signals increases signaling fidelity and specificity as well as ensuring a stringent control of transcription.
the possibility of gα <dig> to bind plcβ and tpr <dig> simultaneously raises some interesting questions regarding the fidelity of g protein signals.
one concern is whether the two signaling pathways can be regulated independently.
stimulation of plcβ by gpcr can be mediated via members of the gαq subfamily  <cit>  or through the gβγ dimer  <cit> , with the latter restricted primarily to some isoforms of plcβ.
like gα <dig>  both gαq  <cit>  and gα <dig>  can interact with tpr <dig>  since many ligands that act on gq-coupled receptors are mitogenic  <cit> , linkage through gαq/tpr <dig> provides a means for the efficient stimulation of the ras/raf/mek/erk cascade for cell proliferation.
given that both plcβ and tpr <dig> are ubiquitously expressed, the ability of gα <dig> as well as other gαq subfamily members to selectively activate one of the two pathways may have to rely on alternative means of signal segregation, such as spatial orientation  <cit> , formation of macromolecular signaling complexes  <cit> , and compartmentalization of signaling components  <cit> .
attachment of the gαq subunit to the lipid bilayer  <cit> , as well as its targeting to plasma membrane microdomains and to intracellular organelles  <cit> , have been shown to affect gαq signaling.
it should also be noted that the presence of p63rhogef can affect the binding of plcβ and tpr <dig> to gα <dig> and differentially inhibit their signaling  <cit> .
the p63rhogef-interacting domain on gα <dig> has yet to be elucidated, but is expected to encompass the α2-β4-α3-β <dig> regions and the c-terminal α <dig> helix based on the crystal structure of gαq-p63rhogef-rhoa complex complex   <cit> .
in this regard, the gα16/z chimeras represent useful tools to confirm such a prediction.
CONCLUSIONS
this study provided evidence for the importance of the β <dig> strand of gα <dig> for the interaction with tpr <dig> and subsequent activation of ras, but the β <dig> strand appears to be dispensable for plcβ interaction.
the integrities of both helical and gtpase domains are essential for plcβ activation.
gα <dig> can signal through tpr1/ras and plcβ simultaneously and independently to regulate transcriptional events involving stat <dig> and nfκb by utilizing different structural domains to bind tpr <dig> and plcβ.
overall, gα <dig> is able to interact with multiple molecular partners to convey different streams of signal transduction.
list of abbreviations used
a1r: type  <dig> adenosine receptor; ags: activator of g protein signaling; cha: n6-cyclohexyladenosine; fbs: fetal bovine serum; g protein: heterotrimeric guanine nucleotide-binding proteins; gpcr: g protein-coupled receptor; gtpase: gtp hydrolase; ikk: inhibitor of κb kinase; ip3: inositol trisphosphates; nfκb: nuclear factor κb; plcβ: phospholipase cβ; ptx: pertussis toxin; rgs: regulator of g protein signaling; stat3: signal transducer and activator of transcription 3; tpr1: tetratricopeptide repeat 1
authors' contributions
amfl and rkhl performed most biological experiments.
exg assisted in experiments.
mkch performed homology modeling.
amfl, mkch and yhw conceived and designed the experiments and wrote the manuscript.
rdy participated in the design of the study.
all authors read and approved the final manuscript.
acknowledgements and funding
we thank cecilia wong and joy chan for helpful discussions.
this work was supported in part by grants from the research grants council of hong kong , the university grants committee , and the hong kong jockey club.
yhw was a recipient of the croucher senior research fellowship.

 heterotrimeric guanine nucleotide-binding proteins are multifaceted signaling modules that relay extracellular signals detected by g protein-coupled receptors to intracellular effector. at the core of the g protein signal transduction machinery is the gα subunit, a gtpase which acts as a timer to limit the activation signal. in the classical g protein activation cycle, the gα subunit needs to associate with the gβγ dimer, the gpcr, and effectors separately or simultaneously at different stages of the cycle. a variety of accessory proteins are now known to modulate the fidelity of the g protein signal. they include regulators of g protein signaling, activators of g protein signaling, and adaptor proteins such as tetratricopeptide repeat. these additional components allow for rapid inactivation or receptor-independent activation of the gα subunit, as well as signal diversification. the large number of different types of binding partners for the gα subunit requires optimal utilization of structural domains that are available for protein-protein interactions. given that gα subunits are typically less than kda in size and are attached to the inner leaflet of the plasma membrane, the binding surfaces available for interaction are limited. nature has partially resolved this constraint by generating different conformations of the gα subunit through the binding and hydrolysis of gtp. the resolution of the crystal structures of several gα subunits in their gdp- or gtp-bound states and as complexes with the gβγ dimer or other interacting proteins have provided valuable insight into the molecular mechanisms of g protein signal transduction. structurally, the gα subunit can be broadly divided into the gtp hydrolase and helical domains with the former harboring the gtp-binding pocket. several regions spreading across the gtpase and helical domains exhibit profound conformational changes when the gα subunit shifts between the gdp- and gtp-bound states. changes in the switch regions provide the molecular basis of g protein activation and effector regulation. in the gtp-bound active state, the gα subunit releases the gβγ dimer and thus allows effectors to bind to the newly exposed surfaces such as the switch ii region. this simplistic view, however, cannot accommodate the increasing numbers of gα-interacting proteins. the activated gα subunit is a preferred partner for multiple effectors, adaptors, and rgs proteins. a central question is whether an activated gα subunit can concurrently regulate multiple signaling pathways by simultaneously binding to different partners in much the same way as an inactive gα subunit forms a complex with the gβγ dimer and the receptor. among the different subfamilies of gα subunits, members of the gαq subfamily have the capacity to activate phospholipase cβ as well as interact with the guanine nucleotide exchange factor p63rhogef, g protein-coupled receptor kinase grk, adaptor proteins such as tpr, and several rgs proteins. these molecules bind to overlapping as well as distinct regions on gαq. it is often assumed that the primary signal generated by gq-coupled receptors is the formation of inositol trisphosphates by plcβ, and that the regulation of downstream kinases and transcription factors are consequential to the production of ip and the subsequent ca2+ mobilization. however, recent studies suggest that gα subunits can concurrently regulate multiple signaling pathways. the ability of gα a member of the gαq subfamily, to interact with the adaptor protein tpr has raised some interesting scenarios. gα is primarily expressed in hematopoietic cells and it can regulate multiple signaling pathways. interestingly, tpr can directly interact with ras especially when the latter is activated and it appears to link gα to ras activation. ras is a small gtpase which acts as a molecular switch for linking various cell surface receptors to intracellular signaling pathways, resulting in cell proliferation and differentiation. we have recently demonstrated that constitutively active gα induces the phosphorylations of transcription factors, such as signal transducer and activator of transcription and nuclear factor κb, through plcβ and ras/raf-1/mek/erk signaling cascades in human embryonic kidney cells. however, it is not known if the binding of tpr to gα affects plcβ signaling and whether tpr and plcβ utilize the same docking site on gα although gα has been shown to interact with the c-terminus of tpr, the structural requirement for gα to interact with tpr has yet to be defined. in the present study, we examined the structural domain of gα for interacting with tpr and assessed whether the same domain is responsible for regulating plcβ. methods materials the human cdnas of gα gα16ql, gαz and gαzql were obtained from missouri s&t cdna resource center. c and c cdnas were previously constructed and characterized. cell culture reagents, including lipofectamine™ and plus™ reagents, and accuprime™ pfx supermix were purchased from invitrogen. anti-gα and anti-gαz were obtained from gramsch laboratories. anti-gα was purchased from torrey pines biolabs. anti-gαz and plcβ antibodies were from santa cruz biotechnology. unbound and affinity gel-conjugated anti-flag antibody were from sigma-aldrich. other antibodies were purchased from cell signaling technology. protein g-agarose and protein cross-linking agent dithiobis were from pierce biotechnology. ecl kit was from ge healthcare bio-sciences. ras activation kit was a product of millipore. construction of chimeras gα chimeras were constructed from the cdnas encoding the human gα and gαz by using pcr techniques. the n-terminal and residues of gα were substituted by the corresponding regions of gαz to generate n n n n n n n and n chimeras, respectively. primer pairs were designed to cover the overlapping regions in forward and reversed directions. for each construct, the 5' fragment was generated with the reversed and t primers, whereas the 3' fragment was made with the forward and sp primer. the two half-products were then annealed together to generate a full-length fragment by another round of pcr using t and sp primers. mirror-images of these constructs were generated analogously and were named c c c c and c chimeras. primers for chimera construction are listed in table pcr was carried out using accuprime™ pfx supermix. the n200-c and n188-c chimeras were constructed using c as the initial template for the 3' half-products and those primers designed for n and n respectively. likewise, zβ and zβ2β were constructed using the c and c primers with n as the initial templates. all chimeras were checked by restriction mapping and then subcloned into pcdna at hind iii and xho i sites. the constructs were fully sequenced by dideoxynucleotide sequencing to confirm the identities. bold and italic nucleotides denote the gα16-derived sequences. cell culture and co-immunoprecipitation experiments hek cells were obtained from the american type culture collection. they were maintained in eagle's minimum essential medium at 5% co 37°c with 10% fetal bovine serum, units/ml penicillin and μg/ml streptomycin. hek cells were grown to 80% confluency in 100-mm tissue culture plates and then co-transfected with ng gα and ng flag-tpr cdnas using μl plus™ and lipofectamine™ reagents in opti-mem™. fbs was replenished h after transfection. cross-linking was performed one day after transfection. transfected cells were washed with pbs twice and then treated with mm dithiobis in pbs for min at room temperature. cells were then washed again with pbs and maintained in quenching solution for min. subsequently, cells were lysed in ice-cold ripa buffer. cell lysates were gently rocked with an anti-gα anti-gαz or anti-plcβ antiserum at 4°c overnight, and then incubated in μl protein g-agarose at 4°c for h. alternatively, the cell lysates were incubated in μl anti-flag affinity agarose gel at 4°c overnight. immunoprecipitates were washed with ice-cold ripa buffer for four times, resuspended in μl ripa buffer and μl × sample buffer and then boiled for min. gα gαz, plcβ and flag-tpr proteins in the immunoprecipitates were analyzed by western blots. ras activation assay hek cells were co-transfected with ng gα, ng flag-tpr and ng ras cdnas. after day, transfectants were serum-deprived for h. cells were then washed twice with ice-cold pbs and lysed with the × mg2+ lysis buffer. cell lysates were pre-cleaned with protein g-agarose and activated ras was immunoprecipitated with μl raf- ras-binding domain-conjugated agarose for min and followed by three washes of μl ice-cold mlb. immunoprecipitates was finally reconstituted in μl mlb and μl × sample buffer and resolved in sds-page for detecting ras using specific antibody. assays for phosphorylated erk, ikk and stat3 hek cells were seeded on a 6-well plate at × cells/well day prior to transfection. transfection was performed with ng gα and ng flag-tpr cdnas using μl plus™ and lipofectamine™ reagents in opti-mem™. the transfectants were serum-deprived overnight one day after transfection. cells were treated with pertussis toxin and n6-cyclohexyladenosine where appropriate, lysed, and then assayed for phosphorylation statuses of erk, stat and ikk as described previously. inositol trisphosphate accumulation assay hek cells were seeded on a 12-well plate at × cells/well one day prior to transfection. cells were transfected with ng gα with or without ng type adenosine receptor cdnas using μl lipofectamine™ reagent in opti-mem™ containing 5% fbs. transfectants were labeled with μci/ml myo-inositol and subsequently assayed for cha-induced ip formation as described previously. nfκb-driven and stat3-driven luciferase assays for stat3-driven luciferase assay, hek cells were seeded on a 96-well microplate at cells/well one day before transfection. cells were transfected with ng gα and ng pstat3-luc luciferase reporter using μl plus™ and lipofectamine™ reagents in opti-mem™ and fbs was replenished after h. when a1r-induced signals were analyzed, ng receptor cdna was included in the transfection. for nfκb-driven luciferase assay, hek cells stably transfected with pnfκb-luc luciferase reporter were seeded on a 96-well microplate at cells/well. the setup and transfection were as described previously. one day after transfection, transfectants were serum-deprived for h and ptx was added where necessary. cells were challenged with or without μm cha for h before measuring the luciferase activity as reported previously. western blotting analysis protein samples were resolved on 12% sds-page and transferred to nitrocellulose membrane. resolved proteins were detected by their specific primary antibodies and horseradish peroxidase-conjugated secondary antisera. the immunoblots were visualized by chemiluminescence with the ecl kit from ge healthcare bio-sciences, and the images detected in x-ray films were quantified by densitometric scanning using the eagle eye ii gel documentation system. molecular modeling of gα subunits gαq in the complex with p63rhogef and rhoa was employed for the illustration of functional domains of gαq, and for creating a molecular model of gα by homologous modeling using swiss-model, which allowed manual adjustments to the alignment of the sequences of gα with gαq in order to accommodate the extraordinarily long α4/β loop of gα molecular models of gαz with or without the mutations at β2/β loop were generated using 3d-jigsaw based on the crystal structure of gαi bound to alf4- and gdp as selected by the default automatic mode. 3d-jigsaw-generated models showed greater dynamics in the loop structures and allowed for the exploration of potential conformational variations caused by the mutations. visualization of various structures was accomplished using ucsf chimera.  extreme termini of gα are not involved in tpr interaction unlike the receptor-interacting domain which is composed of five distinct structures, no discrete localization for effector interaction is generally applicable to the gα subunits, probably because the different signaling pathways entail a diverse spectrum of effector molecules. the gα regions responsible for effector interaction have not been mapped, but there is evidence to suggest that the switch iii and the α helix may participate in the binding of p63rhogef and that the plcβ-interacting domain on gαq mainly encompasses the α2-β4-α3-β regions. the involvement of β strand, α and α helices in the gαq/plcβ complex formation has been revealed clearly in the very recently resolved crystal structure which also confirmed that plcβ can serve as a gap for gαq. in order to identify the structural domains on gα that interact with tpr gα sequences were progressively replaced by those of gαz because the latter does not recognize tpr. chimeras composed of gα and gαz residues were preferred because they are structurally viable. a series of chimeras was made by swapping discrete regions between gα and gαz. construction of the chimeras was guided by the predicted tertiary structure of the gα subunits as well as by our previous experience in determining the receptor and effector interacting domains of gα and gαz. for chimeras with substitutions at either the n- or c-terminus, they were named with a single letter followed by the numbers of amino acids of gαz that substituted the corresponding regions of gα the two chimeras containing either β or β2-β strands of gαz were named with the a letter "z" followed by "β2" or "β2β3", respectively, in order to distinguish them from the descriptions of specific β strand structures. we first examined two chimeras of gα containing either or amino acids of gαz at the c-terminus; representing changes in the α helix alone and α4/β loop plus the α helix, respectively. these two chimeras, named c and c respectively, have been previously constructed and characterized. both chimeras exhibit enhanced coupling to gi-coupled receptors and possess the ability to stimulate plcβ. hek cells were co-transfected with flag-tpr in combination with pcdna gα c or c as illustrated in figure 2c, gα c and c were co-immunoprecipitated with the anti-flag affinity gel with similar levels of flag-tpr moreover, flag-tpr was coimmunoprecipitated along with gα c or c. gα c c and flag-tpr were expressed at detectable and comparable levels in the transfectants. since both chimeras retained the ability to interact with flag-tpr it implies that the c-terminal β and α regions of gα are not required for the interaction with tpr the n-terminus of gα subunits participates in membrane attachment, gβγ binding as well as receptor recognition. another previously constructed chimera, n, was employed to test the possible involvement of the n-terminus of gα in tpr1-binding. the αn helix of n is composed of gαz sequence. as shown in figure 2d, both gα and n co-immunoprecipitated with flag-tpr and similar levels of flag-immunoreactivity were observed. similarly, flag-tpr co-immunoprecipitated with gα and n and the levels of these gα subunits in the immunoprecipitates were essentially the same. like gα16ql, constitutively active n30ql was more readily associated with tpr and this effect was not due to variations in expression levels. given that n still possessed the ability to interact with flag-tpr our results suggest that the region which is critical for direct or indirect binding of tpr may lie between the β strand and α helix of gα the β region of gα interacts with tpr1 to further examine the structural domain of gα for tpr interaction, additional chimeras were constructed by replacing different regions of gα with those of gαz. four different chimeras named n n n and n were thus constructed by replacing a gαz backbone with gα sequences starting from the α α β and α regions, respectively. however, none of the four gα chimeras could be pulled down by anti-flag affinity gel. similarly, flag-tpr could not be co-immunoprecipitated by anti-gα antiserum in chimera-expressing cells despite detectable expression levels of flag-tpr and the chimeras in the cell lysates. in control experiments, both gα and gα16ql were co-immunoprecipitated with flag-tpr. these findings demonstrate that the tpr1-interacting domain must reside between residues and that represent the regions from β to β of gα two more chimeras were constructed to map the tpr1-interacting domain within the first 30- residues. chimeras n and n were made by replacing the n-terminus of gα with gαz sequences up to and including the αa and αd regions, respectively. both n and n were found to co-immunoprecipitate with flag-tpr. association of these two chimeras with flag-tpr was confirmed by reverse co-immunoprecipitation using the anti-gα antiserum. these results narrowed down the tpr1-interacting domain of gα to be among the αe to β regions. before pinpointing the precise location of the tpr1-interacting site on gα we first confirmed the preceding observations with additional chimeras that represent the mirror images of several tested chimeras. chimeras c c and c were essentially the mirror images of n n and n respectively, and they should exhibit phenotypes opposite to those of their counterparts. indeed, c and c could not associate with flag-tpr even though they were efficiently expressed. conversely, chimera c should be able to interact with flag-tpr because its mirror image failed to associate with flag-tpr1; co-immunoprecipitation of c and flag-tpr confirmed their association. these results again indicate that the tpr1-interacting domain of gα lies within the αe-αf-β2-β regions which are common in n n and c but missing in their corresponding mirror images. to tease out the tpr1-interacting domain, two new constructs were made by dissecting the αe-αf-β2-β regions into two halves. c contained the n-terminal half of gα up to and including the αe-αf regions, while n represented its mirror image and contained gα16-specific sequence from the β2-β regions onward. because these two mirror images contained completely different regions of gα and gαz, two distinct anti-gα antisera, targeting either the extreme n-terminus or c-terminus, were required in the co-immunoprecipitation assay. although both mutants were expressed to comparable levels, only n was co-immunoprecipitated with flag-tpr. for the reverse co-immunoprecipations, n and n188ql, but not c and its mutant, were able to interact with tpr. since these findings suggest that only β2-β within the αe-αf-β2-β region is responsible for tpr association, two more chimeras were created to split this region into two halves; c possessed gα16-specific β region whereas n harbored the gα16-specific β region. co-immunoprecipitation experiments with flag-tpr illustrated that n and its constitutively active mutant n200ql interacted with flag-tpr while c and c174ql were ineffective. reverse co-immunoprecipitations with anti-gα antisera confirmed the ability of tpr to associate with n and n200ql. in order to validate the importance of the β region of gα for tpr interaction, n188-c and n200-c were constructed as shown in figure 6a. n188-c is primarily a gαz backbone with the β and β regions from gα while n200-c is essentially gαz with the β region made up of the gα sequence. co-immunoprecipitation experiments using anti-flag or anti-gαz antisera showed that both chimeras and their respective constitutively active mutants interacted with flag-tpr. association of n200-c with flag-tpr suggests that the β region of gα alone is sufficient to confer upon gαz the ability to interact with tpr on the other hand, replacement of the β region of gα with the cognate sequence of gαz is expected to disrupt gα16/tpr interaction. two additional chimeras, named zβ and zβ2β with either the β or β2-β regions of gαz inserted into a gα backbone were constructed to test this hypothesis. zβ2β and zβ2β3ql failed to co-immunoprecipitate with tpr, thus demonstrating the importance of the β and β regions of gα for interaction with tpr however, very weak but detectable associations of zβ and zβ3ql with flag-tpr were observed in co-immunoprecipitation assays. these results confirm the crucial role of the β region for the gα16/tpr interaction, and further suggest that the β region may facilitate the actions of the β strand. activation of ras via the association of tpr with gα and its chimeras association of tpr with ras may provide a more direct link for gα to activate the erk cascade instead of going through the plcβ/pkc pathway. if the β region of gα is essential for functional interaction with tpr and ras, chimeras containing this region should facilitate the activation of ras while those lacking this region ought to be inactive. as predicted, transfectants expressing n200ql exhibited elevated ras activity whereas c174ql did not activate ras. to extend this study, other tpr1-interacting chimeras were evaluated. no significant ras activation was detected when wild-type n n n and c were overexpressed. upon the introduction of the ql mutants of these chimeras, all chimeras induced detectable ras activation as compared to their corresponding wild-type counterparts. likewise, n188-c and n200-c should be capable of activating ras because they possess the β region of gα indeed, the constitutive active mutants of these chimeras activated ras. conversely, zβ2β and zβ did not stimulate the ras activity. these results indicate that chimeras containing the gα16-specific β region possess the ability to activate ras, and such activity is dependent on the gtp-bound conformation of the chimeras. gα16ql-induced ras activation is independent of plcβ signaling the plcβ-interacting domain of gαq was initially mapped to residues 217-, corresponding to the α2-β4-α3-β regions of gα recent resolution of the crystal structure of gαq-plcβ has refined the plcβ-interaction surface on gαq to encompass mainly β strand, α and α helices. since the putative plcβ-interacting domain of gα is in proximity of the tpr1-interacting β region, we asked if activation of ras and plcβ can occur independently. the chimeras were transiently expressed in the absence or presence of the gi-coupled a1r and then assayed for ip formation with or without μm cha. predictably, cha-induced ip formation was observed with transfectants co-expressing gα n c or c. chimeras with parts of the plcβ-interacting regions of gα replaced by cognate gαz sequences should exhibit impaired ability to regulate plcβ. such chimeras include n n n c c c c c n188-c and n200-c indeed, all ten chimeras failed to stimulate plcβ in response to cha, although cha was capable of inhibiting camp formation in the transfectants. the putative plcβ-interacting region of gα is intact in n n n n n zβ2β and zβ and hence these chimeras are expected to support a1r-mediated ip formation. however, only transfectants harboring n zβ2β or zβ responded to cha with a significant increase in ip formation. the lack of response to cha challenge may be attributed to impairment in receptor/g protein recognition. to exclude such a possibility, we tested the ability of the constitutively active mutants of the chimeras to stimulate plcβ. those mutant chimeras with the plcβ-interacting region disrupted or replaced by gαz residues did not exhibit any stimulation of plcβ, while gα16ql and the previously characterized mutants such as c efficiently induced ip formation in the transfectants. among the mutant chimeras with the α2-β4-α3-β region intact, only n188ql, n200ql, zβ2β3ql and zβ3ql constitutively stimulated the plcβ activity. the constitutive activity of n200ql suggests that the inability of n to mediate a1r-induced stimulation of plcβ may be attributed to defective recognition of receptor. with the exception of n n and n the plcβ-stimulating abilities of the chimeras were in general agreement with the predicted presence of the putative plcβ-interacting region. hek cells were co-transfected with a1r and pcdna or the indicated gα subunit. transfectants were labeled with inositol in dmem containing 5% fbs overnight. ip formations were examined in the absence or presence of μm cha for min. fold stimulations were calculated as the ratios of cha-induced to basal ip accumulations or ql-induced to wild-type ip accumulations. data represent the mean ± s.d. of triplicate determinations of a single representative experiment, n = 3; significant responses are shown in bold and italic. constructs containing an intact putative plcβ binding domain of gα are marked with a "yes". * cha-stimulated ip production is significantly greater than basal; paired t-test, p ≤. # ql-stimulated ip production is significantly greater than the corresponding wild-type activity; paired t-test, p ≤. a ql-induced fold stimulation of 16z was extrapolated from. to confirm that the plcβ-interacting regions of gα16-interaction are distinct from that of tpr two series of gα chimeras were tested. the first series contained chimeras harboring the putative plcβ-interacting domains, including n n and n hek cells were cotransfected with plcβ and gα or a chimera and then subjected to co-immuniprecipitation using antisera against either gα or plcβ interaction between plcβ and gα was clearly evident, with gα16ql generating a stronger interaction with plcβ. chimeras n n and n were all capable of being co-immunoprecipitated with plcβ another set of chimeras including c c and c was similarly tested. due to the replacement of the putative plcβ-interacting regions with sequences of gαz, these chimeras were expected not to interact with plcβ indeed, even with successful expression of the chimeras and plcβ no plcβ interaction was detectable for these chimeras. these results support the notion that the β region of gα is dispensable for the interaction with plcβ. since several chimeras can apparently activate ras through tpr despite their inability to stimulate plcβ, they represent useful tools in delineating complex signaling networks such as those for the regulation of stat and nfκb. if these chimeras can induce the phosphorylation and activation of stat and nfκb, then it would imply that plcβ activity is not essential. conversely, a lack of activity on stat and nfκb by these chimeras would indicate that plcβ action is required. transfectants co-expressing the chimeras and a1r were stimulated with cha, and the phosphorylation of stat and inhibitor of κb kinase monitored by western blot analysis using phosphoprotein-specific antibodies. as shown in table chimeras with dual tpr1/ras and plcβ activating capabilities effectively mediated cha-induced stat and ikk phosphorylations, with magnitudes similar to those of gα these chimeras included n n c c zβ2β and zβ n failed to mediate the cha-induced stat phosphorylation but supported the ikk phosphorylation. tpr1-interacting chimeras which lack plcβ activity were unable to support cha-induced phosphorylation of stat and ikk. because erk is required for gα16-mediated stat and nfκb activation, we further tested the ability of the chimeras to mediate erk phosphorylation. the profile of erk phosphorylation mediated by the chimeras closely resembled those for stat and ikk. chimeras lacking both plcβ and tpr1/ras activities, including n n n n c c c and c did not support any of the cha-induced responses, whereas n n n c c zβ2β and zβ mediated cha-induced erk phosphorylation. hek cells were co-transfected with adenosine a1r and pcdna or the indicated gα subunit. transfectants were serum starved overnight in the presence of ng/ml of ptx and then challenged with μm cha for min. cell lysates were then resolved and immunoblotted against various specific antibodies. results are expressed as fold stimulation over the basal, n = 3; significant responses are shown in bold and italic. constructs shown to co-immunoprecipitate with flag-tpr or stimulate plcβ activity are marked with a "yes". * cha-stimulated phosphorylation of the target protein is significantly greater than the basal; paired t-test, p ≤. a significant plcβ activation observed with the ql mutant only. b very weak association with tpr was detected in co-immunoprecipitation assays. finally, we employed luciferase reporter gene assays to demonstrate transcriptional regulation of stat and nfκb by those chimeras that possess dual tpr1/ras and plcβ activating capabilities. in agreement with our previous studies, cha induced stat3- and nfκb-driven luciferase activities in transfectants co-expressing gα but not gαz. n n n c c and zβ chimeras all supported cha-induced stat3- and nfκb-driven luciferase activities, whereas no transcriptional activation was observed in transfectants co-expressing chimeras that failed to mediate stat ikk, and erk phosphorylations. collectively, these studies demonstrate that dual tpr1/ras and plcβ activating capabilities of gα may be essential for its regulation of complex signaling networks such as those for the activation of stat and nfκb. hek cells were co-transfected with adenosine a1r, pstat3-luc/pnfκb-luc, and pcdna or the indicated gα subunit. transfectants were serum starved for h in the presence of ng/ml of ptx and then challenged with μm cha overnight. cell lysates were analyzed for luciferase activity. data represent the mean ± s.d. of triplicate determinations, n = 3; significant stimulations are shown in bold and italic. * cha-stimulated protein phosphorylation is significantly greater than the basal; paired t-test, p ≤. discussion protein-protein interactions are central to the functions of gα subunits and each gα subunit has to coordinate such interactions in a timely manner according to its guanine nucleotide binding state. once activated, the gtp-bound gα subunit has a limited time-span to regulate downstream effectors before its intrinsic gtpase activity turns it back to the inactive gdp-bound state, a process which can occur rapidly in the presence of rgs proteins. hence, simultaneous regulation of multiple signaling pathways by activated gα is desirable but this entails the deployment of different binding surfaces. although ras can be activated indirectly by plcβ signaling, our results provide a structural basis for gα to stimulate ras through interaction with tpr instead of plcβ. moreover, functional analyses of gα16/z chimeras reveal that dual stimulation of tpr1/ras and plcβ may be essential for gα to activate downstream transcription factors such as stat and nfκb. the extensive array of gα16/z chimeras has enabled us to pinpoint the tpr1-interacting domain to the β region of gα the β2-β region lies between the switch i and ii regions and is accessible for protein-protein interaction. since activation of the gα subunit alters the conformation of several switch regions drastically, proteins like plcβ that bind to the switch regions will interact with the gtp-bound active state much effectively than the gdp-bound basal state of the gα subunit. however, the differences of the interactions between tpr and gα16wt or gα16ql are comparably smaller, and tpr can obviously bind to gα16wt. such observation implies that tpr may bind to gα at regions with relatively less drastic conformational changes. our results suggested that the β strand of gα alone appears to be sufficient for the interaction with tpr the mere incorporation of the β region of gα confers upon gαz the ability to bind tpr. n200-c has the amino acid sequence 201ktnlrivdvg from gα inserted into a gαz backbone, but only the first six residues are different from the corresponding gαz sequence. based on the crystal structure of gαi, molecular modeling of gαz reveals that glu is located at the "hook-shaped" β2-β turn, which is often composed of two oppositely charged residues flanked by two interacting hydrophobic residues except gα with a threonine following the lys the predicted β2-β turn of n200-c showed a more widened conformation, which presumably forms a characteristic microdomain on gα for tpr interaction. the fact that the chimera zβ exhibited residual binding to tpr suggested that other regions like the β strand may also participate in the binding of tpr because its removal in the zβ2β chimera can further suppress tpr binding as compared to zβ. since several chimeras failed to generate a response in all of the functional assays, these chimeras might not be able to adopt the active conformation properly and it remains possible that additional residues other than the β region may bind to tpr although the chimeras used in the present study were not tailored for the mapping of the plcβ-interacting domain of gα they nonetheless proved useful in locating the overall site for the binding of plcβ. as in the case of gαq, the regions comprising of β β α and α are likely to form the plcβ-interacting surface of gα because chimeras with one or more of these regions disrupted all failed to stimulate ip formation. a more interesting observation is that chimeras including n n n and n could not activate plcβ at all, and only the constitutively active form of n could stimulate plcβ weakly, whereas the chimera n recapitulates the plcβ-activating capability. n and n contained all the putative plcβ-interacting domains and yet could not activate plcβ, albeit both interacted with tpr and activated ras. one plausible explanation is that both chimeras have a hybrid helical domain which may affect its structural and functional integrity. indeed, all gα16/z chimeras that can stimulate plcβ activity contain a contiguous helical core in addition to the putative plcβ-interacting domains. an early study of gαs suggested that its helical domain could function as an internal gap for the gtpase domain, and the intra-molecular interaction between the gtpase and helical domains was proven to be essential for guanine nucleotide binding and receptor-mediated activation. furthermore, the αc-αd loop of gα subunits has been recognized as switch iv which shows significant conformational changes in different guanine nucleotide-binding states and could reduce the nucleotide exchange rate when mutated. it is therefore possible that a hybrid helical domain may impede the activation of plcβ by the gα16/z chimeras. the sudden regain of plcβ-activating property of n which contains a helical domain completely derived from gαz also implies that structural integrity of the helical domain is critical for plcβ activation. as revealed in the very recent crystal structure of gαq-plcβ, the β and β strands of gαq interact extensively with the c domain of plcβ and these two strands also heavily contribute to the overall scaffold of the gtpase domain. replacement of one or both of them, as in the n and n chimeras, might disrupt plcβ interaction severely. nonetheless, n can successfully form a heterotrimer with flag-tagged gβγ dimer in co-immunoprecipitation experiment, excluding the possibility of improper folding of the chimera. a molecular model of gα is constructed by homologous modeling based on the crystal structure of gαq to visualize the potential interacting surfaces for plcβ and tpr except for the unusually long α4-β loop and differences in the n-terminal helix, gα basically fit very well to the structure of gαq. assuming that gα utilizes domains similar to those of gαq for the binding of plcβ, it is entirely feasible for gα to simultaneously regulate plcβ and tpr as shown in the right panel of figure 9a, the critical plcβ-interacting residues are clustered mostly on the left hand side of the gtpase domain of gα, while the putative tpr1-interacting β region is located in the lower quadrant of the right hand side without severe overlap with the plcβ-interacting region. preliminary co-immunoprecipitation studies indeed suggest the existence of gα16/plcβ and plcβ/tpr complexes. further studies will be required to confirm if a gα16/plcβ/tpr complex truly exists. tpr motif-containing adaptors such as rapsyn are known to cluster signaling molecules for the efficient propagation of signals. it is conceivable that tpr may serve a similar function in g protein pathways. its lack of association with gα gαt and gαz suggests that tpr can selectively link g protein signals to ras-dependent pathways. for those that can interact with tpr it remains to be determined if binding to tpr confers upon them the same repertoire of signaling capabilities. the distinct locations for plcβ- and tpr1-interacting regions infer that gα can regulate them independently. indeed, we have demonstrated that some chimeras can bind tpr and induce ras activation even though they lack the ability to stimulate plcβ. this raises an interesting possibility that, depending on the composition of the signaling modules within a cell, activation of gα may differentially regulate tpr1/ras and plcβ signaling pathways. both ras and plcβ activities are apparently required for the regulation of stat and nfκb, but it is not clear if the two components are arranged in parallel or in series. given that the activation of stat and nfκb could only be detected with chimeras possessing the ability to activate both ras and plcβ, it would seem that the two pathways are independently required for the regulation of stat and nfκb. the need for multiple input signals increases signaling fidelity and specificity as well as ensuring a stringent control of transcription. the possibility of gα to bind plcβ and tpr simultaneously raises some interesting questions regarding the fidelity of g protein signals. one concern is whether the two signaling pathways can be regulated independently. stimulation of plcβ by gpcr can be mediated via members of the gαq subfamily or through the gβγ dimer, with the latter restricted primarily to some isoforms of plcβ. like gα both gαq and gα can interact with tpr since many ligands that act on gq-coupled receptors are mitogenic, linkage through gαq/tpr provides a means for the efficient stimulation of the ras/raf/mek/erk cascade for cell proliferation. given that both plcβ and tpr are ubiquitously expressed, the ability of gα as well as other gαq subfamily members to selectively activate one of the two pathways may have to rely on alternative means of signal segregation, such as spatial orientation, formation of macromolecular signaling complexes, and compartmentalization of signaling components. attachment of the gαq subunit to the lipid bilayer, as well as its targeting to plasma membrane microdomains and to intracellular organelles, have been shown to affect gαq signaling. it should also be noted that the presence of p63rhogef can affect the binding of plcβ and tpr to gα and differentially inhibit their signaling. the p63rhogef-interacting domain on gα has yet to be elucidated, but is expected to encompass the α2-β4-α3-β regions and the c-terminal α helix based on the crystal structure of gαq-p63rhogef-rhoa complex complex. in this regard, the gα16/z chimeras represent useful tools to confirm such a prediction.  this study provided evidence for the importance of the β strand of gα for the interaction with tpr and subsequent activation of ras, but the β strand appears to be dispensable for plcβ interaction. the integrities of both helical and gtpase domains are essential for plcβ activation. gα can signal through tpr1/ras and plcβ simultaneously and independently to regulate transcriptional events involving stat and nfκb by utilizing different structural domains to bind tpr and plcβ. overall, gα is able to interact with multiple molecular partners to convey different streams of signal transduction. list of abbreviations used a1r: type adenosine receptor; ags: activator of g protein signaling; cha: n6-cyclohexyladenosine; fbs: fetal bovine serum; g protein: heterotrimeric guanine nucleotide-binding proteins; gpcr: g protein-coupled receptor; gtpase: gtp hydrolase; ikk: inhibitor of κb kinase; ip3: inositol trisphosphates; nfκb: nuclear factor κb; plcβ: phospholipase cβ; ptx: pertussis toxin; rgs: regulator of g protein signaling; stat3: signal transducer and activator of transcription 3; tpr1: tetratricopeptide repeat 1 authors' contributions amfl and rkhl performed most biological experiments. exg assisted in experiments. mkch performed homology modeling. amfl, mkch and yhw conceived and designed the experiments and wrote the manuscript. rdy participated in the design of the study. all authors read and approved the final manuscript. acknowledgements and funding we thank cecilia wong and joy chan for helpful discussions. this work was supported in part by grants from the research grants council of hong kong, the university grants committee, and the hong kong jockey club. yhw was a recipient of the croucher senior research fellowship.
using gα as a model, we asked if its recently discovered adaptor protein tetratricopeptide repeat binds to the same region as its canonical effector, phospholipase cβ. our results suggest that gα can signal through tpr1/ras and plcβ simultaneously and independently. progressive replacement of gα sequence with the corresponding residues of gαz eventually identified a stretch of six amino acids in the β region of gα which are responsible for tpr interaction and the subsequent ras activation. the ability of the chimeras to activate downstream transcription factors such as signal transducer and activator of transcription and nuclear factor κb appeared to be associated with plcβ signaling. since the β region only minimally contributes to interact with plcβ, several chimeras exhibited differential abilities to stimulate plcβ and ras. as the core of the g protein signal transduction machinery, the gα subunits are required to interact with multiple partners. the β region of gα is essential for interaction with tpr and the subsequent activation of ras, but has relatively minor influence on the plcβ interaction. the gtp-bound active state of many gα subunits can bind a multitude of effectors and regulatory proteins. yet it remains unclear if the different proteins utilize distinct or common structural motifs on the gα subunit for binding. gα may utilize different structural domains to bind tpr and plcβ.
 g protein-coupled receptors constitute the largest family of cell surface receptors in the mammalian genome. as the core of the g protein signal transduction machinery, the gα subunits are required to interact with multiple partners. the gtp-bound active state of many gα subunits can bind a multitude of effectors and regulatory proteins. yet it remains unclear if the different proteins utilize distinct or common structural motifs on the gα subunit for binding. using gα as a model, we asked if its recently discovered adaptor protein tetratricopeptide repeat binds to the same region as its canonical effector, phospholipase cβ.  we have examined the specificity of gα16/tpr association by testing a series of chimeras between gα and gαz. tpr co-immunoprecipitated with gα and more tightly with its constitutively active gα16ql, but not gαz. progressive replacement of gα sequence with the corresponding residues of gαz eventually identified a stretch of six amino acids in the β region of gα which are responsible for tpr interaction and the subsequent ras activation. insertion of these six residues into gαz allowed productive tpr1-interaction. since the β region only minimally contributes to interact with plcβ, several chimeras exhibited differential abilities to stimulate plcβ and ras. the ability of the chimeras to activate downstream transcription factors such as signal transducer and activator of transcription and nuclear factor κb appeared to be associated with plcβ signaling.  our results suggest that gα can signal through tpr1/ras and plcβ simultaneously and independently. the β region of gα is essential for interaction with tpr and the subsequent activation of ras, but has relatively minor influence on the plcβ interaction. gα may utilize different structural domains to bind tpr and plcβ. 