g protein chimera, 1 a single gβ subunit locus controls crosstalk

G protein chimera, 1

A single Gβ subunit locus controls crosstalk between PKC and G protein regulation of N-type calcium channels

Clinton J. Doering*, Alexandra E. Kisilevsky*, Zhong-Ping Feng*, Michelle I. Arnot, Jean Peloquin, Jawed Hamid, Wendy Barr, Aparna Nirdosh, Brett Simms, Robert J. Winkfein, and Gerald W. Zamponi Department of Physiology and Biophysics, Cellular and Molecular Neurobiology Research Group, University of Calgary, Calgary, T2N 4N1, Canada. * these authors contributed equally Running title: G protein-PKC crosstalk Address for correspondence: Gerald W. Zamponi, Ph.D. Department of Physiology and Biophysics University of Calgary 3330 Hospital Dr. NW Calgary, T2N 4N1 Canada Tel. (403) 220-8687 Fax. (403) 210-8106 Email. [email protected]

by guest on February 10, 2018http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


Abstract

The modulation of N-type calcium channels is a key factor in the control of neurotransmitter

release. Whereas N-type channels are inhibited by Gβγ subunits in a G protein β isoform

dependent manner, channel activity is typically stimulated by activation of PKC. In addition,

there is crosstalk among these pathways, such that PKC dependent phosphorylation of the Gβγ

target site on the N-type channel antagonizes subsequent G protein inhibition, albeit only for Gβ1

mediated responses. The molecular mechanisms that control this G protein β subunit subtype

specific regulation have not been described. Here, we show that G protein inhibition of N-type

calcium channels is critically dependent on two separate, but adjacent ~20 amino acid regions of

the Gβ subunit, plus a highly conserved Asn-Tyr-Val motif. These regions are distinct from

those implicated previously in Gβγ signaling to other effectors such as GIRK channels,

phospholipase-β2 and adenylyl cyclase, thus raising the possibility that the specificity for G

protein signaling to calcium channels might rely on unique G protein structural determinants. In

addition, we identify a highly specific locus on the Gβ1 subunit that serves as a molecular

detector of PKC dependent phosphorylation of the G protein target site on the N-type channel α1

subunit, thus providing for a molecular basis for G protein-PKC crosstalk. Overall, our results

significantly advance our understanding of the molecular details underlying the integration of G

protein and PKC signaling pathways at the level of the N-type calcium channel α1 subunit.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


Introduction:

The modulation of calcium channels at presynaptic nerve terminals is a key factor in regulating

synaptic efficacy (1,2). It is now well established that the activation of G protein coupled

receptors inhibits presynaptic calcium channel activity, and thus neurotransmitter release (3). G

protein inhibition of both N-type and P/Q-type calcium channels appears to be exclusively

mediated by the G protein βγ subunit (4,5), with the Gβ subunit being the main determinant of

calcium channel inhibition. Putative G protein βγ subunit interactions sites have been identified

within the intracellular loop linking domains I and II of the calcium channel α1 subunit (6-8), as

well as in the carboxy terminus region (9). To date, five different types of G protein β subunits

have been identified and shown to mediate varying effects on native and transiently expressed N-

type calcium channels (10,11). Moreover, N-type and P/Q-type calcium channels appear to be

differentially modulated by different types of G protein β subunits (12), thus providing for a

mechanism by which different G protein coupled receptors may selectively regulate individual

presynaptic calcium channel subtypes.

In contrast, activation of protein kinase C (PKC) results in an upregulation of N-type

channel activity (13,14). There is a complex interplay between PKC and G protein pathways,

such that activation of PKC antagonizes subsequent receptor mediated G protein inhibition of

presynaptic calcium channels (15,16). This effect is mediated via PKC dependent

phosphorylation of a single threonine residue located in the G protein interaction site within the

domain I-II linker region of the N-type calcium channel α1 subunit (17), thus allowing the

channel protein to integrate multiple modulatory inputs. Interestingly, this crosstalk between G

protein and PKC pathways appears to be a selective feature of the G protein β1 subunit, thus

allowing PKC to selectively antagonize G protein inhibition mediated by a subset of (i.e.,


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


predominantly Gβ1 coupled) receptors (18). However, although calcium channel structural

determinants of G protein regulation and PKC crosstalk have received considerable attention (3),

there is relatively scant information that concerns the G protein structural determinants that

underlie N-type channel regulation, and PKC- G protein crosstalk. Alanine mutagenesis of Gβ

residues known to interact with Gα disrupts Gβ coupling to a series of down stream effectors,

including calcium channels, adenylyl cyclase, GIRK channels, and phospholipase-β2 (19,20).

This suggests that there may be partial overlap in the G protein β subunit structural determinants

that control the functional interactions both within the heterotrimeric G protein complex, and

with downstream signalling targets. Ford et al. (19) identified six amino acid residues (Lys-78,

Met-101, Asn-119, Thr-143, Asp-186 and Trp-332) on the Gβ1 subunit that, when mutated to

alanine, reduced the ability of Gβ1 to inhibit N-type calcium channels. In addition, mutagenesis

of two residues (Leu-55 and Ile-80) resulted in an increased ability to regulate N-type channels.

However, these residues are completely conserved across all types of G protein β subunit

subtypes which implies that they cannot account for the differential effects of different types of

Gβ subunits on calcium channel activity. Hence, additional Gβ structural determinants control G

protein modulation of N-type channels.

Here, we utilized chimeric and mutant G protein β subunits to systematically identify G

protein structural determinants that control their action on N-type channels. We identify a

hotspot of amino acid sequences in the Gβ1 subunit that is essential for N-type channel

modulation. In addition, we localize crosstalk behavior to a single locus on the Gβ1 subunit, thus

identifying a molecular switch that allows the G proteins to detect a phosphorylated N-type

calcium channel. In this context, our data close a major gap in our understanding of the


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


complex interplay between G protein and PKC regulation of presynaptic calcium channel

activity.

Materials and Methods

cDNAs

The cDNAs encoding human Gβ1 and Gγ2, rat Gβ5 and EGFP-tagged Gβ1 and Gβ5 subunits were

described by us previously (12,21). Wild type rat calcium channel subunits were donated by

Terry Snutch (University British Columbia), the T422E mutant N-type channel was described

previously (17).

Chimeric constructs

Chimeras were created in two steps. For an initial round of chimeras, MluI, ApaLI, and EagI

sites were inserted into both Gβ1 and Gβ5 encoding cDNAs at exactly complimentary positions,

using the Quik Change™ Site-Directed Mutagensis Kit (Stratagene, La Jolla CA, USA). In each

case, the entire coding region was sequenced after mutagenesis. Together with the 5 prime and 3

prime cloning sites (KpnI and XhoI, respectively) and the presence of additional ApaLI and EagI

sites in the pMT2 vector sequence, this allowed the swapping of four different regions (i.e.,

residue 1-47, 48-168, 168-280, 280-340; numbering according to Gβ1 sequence, see Fig. 1A)

between Gβ1 and Gβ5 through cutting and ligating. Successful creation of the chimeras was

confirmed via sequencing. Note that positions 47,168, 280 and 340 in Gβ1 correspond to

positions 54, 179, 294 and 353 in Gβ5.

A second round of chimeras was created via PCR, using wild type Gβ subunits and the

initial set of chimeras as templates. Briefly, sense and antisense oligonucleotides, spanning the

junctions of the new chimeras, were synthesized. Subsequently, these were used to amplify


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


(with vector upstream and downstream primers) each end of the new chimera, using the

appropriate clone as a template. Overlaps included in the junction spanning oligonucleotides

allowed the two fragments to anneal at those points. The two fragments were gel isolated,

combined in equimolar concentrations and subjected to another round of high fidelity PCR, with

only the upstream and downstream primers included in the reaction mix. Proofstart DNA

polymerase (Qiagen) was used for all PCRs. Purified, full length PCR products were isolated

after restriction digestion and cloned in the appropriate vector. All fragments generated by PCR

were sequenced after cloning to ensure no PCR induced errors were incorporated into the final

clones.

Point mutations in Gβ1 and Gβ3

Point mutations in both Gβ1 and Gβ3 were also created using the Quik Change kit as described

above. The presence of the mutations and absence of mutagenesis errors was determined via

DNA sequencing.

EGFP tagged constructs

N-terminal fluorescently tagged chimeric Gβ proteins were created using CLONTECH (Palo

Alto, CA) Living Colors™ C-terminal Enhanced Green Fluorescent Protein (EGFP) vectors.

Briefly, chimeric G protein β subunits excised from the pMT2-XS vector using previously

engineered restriction sites (XhoI and KpnI found in the 5 prime and 3 prime regions

respectively; (12)), and sub-cloned in frame into the EGFP vectors. Correct insertion within the

EGFP vector was confirmed with both restriction enzyme digestion and sequencing.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


Tissue culture and transient transfection of tsA-201 cells

Human embryonic kidney tsA-201 cells were grown and transfected with calcium phosphate as

described by us previously in detail (21). In each experiment involving calcium channels, wild

type or mutant rat Cav2.2 calcium channel α1 subunits were co-transfected with rat β1b, rat α2-δ1,

Gγ2 and an EGFP expression marker (except in the case where EGFP tagged G proteins were

used), plus one of wild type or chimeric Gβ subunits. For experiments involving GIRK channels,

GIRK1 and GIRK4 subunits were used instead of calcium channel subunits. To prevent cells

from overgrowing, cells are routinely placed in a 28°C incubator 12 hours after transfection.

Under these conditions, tsA-201 cells change their morphology such that they appear rounded

(see Fig. 1C, inset).

Patch clamp recordings and data analysis

Glass cover slips carrying transfected cells were transferred to a 3 cm culture dish containing

recording solution comprised of 20 mM BaCl2, 1 mM MgCl2, 10 mM HEPES, 40 mM

tetraethylammonium chloride (TEA-Cl), 10 mM glucose, 65 mM CsCl, (pH 7.2 with TEA-OH).

Whole cell patch clamp recordings were performed using an Axopatch 200B amplifier (Axon

Instruments, Foster City, CA) linked to a personal computer equipped with pCLAMP v 6.0, 80.

or 9.0. Patch pipettes (Sutter borosilicate glass, BF150-86-15) were pulled using a Sutter P-87

microelectrode puller, fire polished and showed typical resistances of 3 to 4 MΩ. The internal

pipette solution contained 108 mM cesium methanesulfonate, 4 mM MgCl2, 9 mM EGTA, 9 mM

HEPES (pH 7.2). Series resistance was compensated by 80-85%. Leak currents were negligible.

Data were filtered at 1 kHz and recorded directly onto the hard drive of the computer. Unless

stated otherwise, currents were evoked by stepping from –100 mV to a test potential of +20 mV.

G protein inhibition was assessed by application of a strong depolarizing (+150 mV) prepulse


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


(PP) for 50 ms. Typically, only cells with current amplitudes greater than 50 pA were used for

analysis. The degree of prepulse relief of tonic G protein inhibition was determined as the ratio

of peak current amplitudes seen after (I(+PP)) and before (I(-PP)) the prepulse and reflects the

ability of a given G protein β subunit to inhibit N-type current activity. The PP paradigms were

programmed using the “train” and “user list” functions in pCLAMP. For experiments involving

activation of protein kinase C, PMA (Sigma) was dissolved in dimethylsulfoxide at a 1 mM

stock concentration, and diluted into the recording solution at a final concentration of 30 nM.

Control solution, or solution containing PMA was perfused onto cells via a home built gravity

driven microperfusion system.

For recordings involving GIRK channels, GIRK1 and GIRK4 subunits, whole cell

recordings were conducted using an internal solution of 100 mM K-gluconate, 40 mM KCl), 10

mM HEPES, 5 mM EGTA 5, 1 mM MgCl2, and 5 mM NaCl (pH 7.4 with KOH). The external

solution contained 25 mM KCl, 10 mM HEPES, 10 mM glucose, and 116 mM NaCl (pH 7.4

with NaOH). Under these conditions, the predicted reversal potential for potassium is

approximately -30 mV. GIRK channel activity was assessed by holding the cells at –35 mV,

followed by an application of a voltage ramp from –120 mV to +60 mV over 525 ms. Only cells

displaying inward rectification were used for analysis, and whole cell GIRK conductance was

obtained by a linear fit to the inward current. Whole cell capacitance ranged from 5 pF to 40 pF.

In this range, there was no correlation between capacitance and whole cell conductance (r2=0.15,

not shown), and hence, data are plotted in Fig. 4 as whole cell conductance rather than current

densities. For each batch of transfected cells, we determined GIRK activity in the absence of Gβ

subunits.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


All data were analyzed using Clampfit (Axon Instruments) and fitted in Sigmaplot 4.0

(Jandel Scientific). Statistical analysis was carried out in SigmaStat via t-tests, or as appropriate

via ANOVA with a post hoc Tukey test, or for GIRK experiments, ANOVA on the ranks (Dunn

method).

Confocal Microscopy

Imaging was carried out at the Seaman Family MR Research Center Confocal Microscopy and

Imaging Facility. Briefly, tsA-201 cells were transfected with DNA encoding the N-type

calcium channel (Cav2.2 α1, α2-δ1, and β1b), Gγ2 and EGFP-tagged G protein β subunits. Initially

coverslips containing cells of interest were placed in a glass-bottom petri-dish and visualized

with an inverted IX70 Olympus microscope. Confocal images were created using an Olympus

Fluoview Confocal Laser Scanning Miscroscope (confocal aperture 2). Cells were stimulated

with an argon 488 nm laser. Settings were chosen for laser power, PMT gain, and offset so that

pixel densities were just below saturation levels. Transmitted image visualization was cpnducted

with DIC/Nomarski Interference Contrast microscopy (21).


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


Results:

Determinants of N-type channel inhibition by Gβ1 subunits

We have shown previously that rat N-type calcium channels are potently inhibited by Gβ1

subunits, whereas Gβ5 subunits have no significant effect on current activity (Ref. 12; see also

Fig. 1C). To determine key G protein β subunit structural determinants that control calcium

channel inhibition, the differential Gβ1/Gβ5 effects were used as the basis for a chimeric

approach. Initially the Gβ1 and Gβ5 subunits were each divided into four complimentary regions

which were exchanged in various combinations (see Fig. 1A for example). The chimeric

constructs were coexpressed with N-type (Cav2.2+β1b+α2-δ1) calcium channels, the Gγ2 subunit,

and an EGFP selection marker, and tonic G protein inhibition was assessed by application of

strong depolarizing prepulses (PP) as described in the Materials and Methods. As shown in Fig.

1B, Gβ1 mediates a robust tonic inhibition as reflected by a large increase in current amplitude

following the PP. Replacement of the first 47 residues or the last 60 residues of Gβ1 with Gβ5

individually, or in combination, preserved the ability of Gβ1 to inhibit N-type channels, despite

the fact that Gβ1 and Gβ5 share only 30% and 60% sequence identity in these two regions,

respectively. In contrast, as illustrated in Fig. 1C, replacement of either residues 47-168, or

residues 168-280 with corresponding Gβ5 sequence alone, or in combination with other

substitutions, reduced the degree of prepulse relief to that seen in the presence of wild type Gβ5

(note that prepulse relief observed with Gβ5 does not differ significantly from conditions where

no G proteins are coexpressed). To minimize the possibility that the lack of modulation might be

due to lack of expression, or due to inappropriate targeting of the Gβ subunit chimeras, we

generated N-terminally EGFP-tagged versions of the wild type and key chimeric G proteins, and


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


analyzed their subcellular distribution via fluorescence confocal microscopy. As shown in the

inset of Fig. 1C, the wild type and chimeric G protein subunits were detected in the plasma

membrane, suggesting that they are indeed properly expressed and targeted in tsA-201 cells.

Moreover, the observation that all of the Gβ constructs display robust plasma localization implies

that the observed effects are not secondarily due to an inability of certain chimeras to assemble

into Gβγ dimers. In addition, we have shown previously via Western blot and kinetic analyses

that transient transfection of wild type G protein results in saturating levels of Gβ which, in turn,

leads to a homogenous population of G protein bound N-type calcium channels (21). Taken

together, these data suggest that the failure to observe G protein inhibition with certain chimeras

was not simply due a lack of expression/targeting.

Overall, our data obtained with the initial set of chimeras suggest that although the N-

and C-terminal regions of Gβ are not critical determinants of G protein inhibition of N-type

channels, one or more regions between residues 47 and 280 are essential for G protein inhibition.

To further elucidate the G protein β subunit structural determinants of N-type channel

modulation, we created additional chimeras with sequence substitutions in regions 47-168 and

168-280. As shown in Fig. 2, replacing Gβ1 residues 47-116 or 116-168 with corresponding Gβ5

sequence each resulted in loss of G protein inhibition. Substitutions of smaller fragments within

these two regions revealed that replacement of regions 47-75, 75-100, and 116-140 did not block

the ability of Gβ1 to inhibit N-type channel activity. On the contrary, the 47-75 construct

displayed a dramatically enhanced ability to inhibit N-type channel activity. In contrast,

substitution of residues 140-168, or replacement of an Asn-110, Tyr-111, Val-112 motif (that is

highly conserved in all G protein β subunits with the exception of Gβ5) with Gβ5 (i.e., Cys-Ala-

Ile) sequence eliminated the ability of Gβ1 to inhibit N-type calcium channels. A similar analysis


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


for residues 168-280 is shown in Fig. 3. As shown in Figs. 3A and B, substitution of residues

204-248, 248-280 and 168-186 maintained the ability of Gβ1 to inhibit N-type calcium channels,

whereas substitution of residues 186-204 abolished Gβ1 modulation. Taken together, there

appear to be at least three separate regions that are responsible for the differential effect of Gβ1

and Gβ5 on N-type calcium channel activity. It is unlikely that these effects would have arisen

from a global disruption of Gβ1 subunit folding due to the presence of Gβ5 sequence, since Gβ1

activity was retained for a majority of the Gβ5 substitutions that were created (i.e., 1-47, 47-75,

75-100, 116-140, 168-186, 204-248, 248-280). To ensure that the effects of the chimeras were

specific rather than due to inappropriate folding, we first attempted to create a gain of function

chimera in which regions in Gβ5 were concomitantly replaced with corresponding Gβ1 residues

110-112, 140-168, and 186-204. However, the Gβ5(110-112, 140-168, 186-204) chimera did not

result in significant G protein inhibition (I+PP/I-PP=1.11±0.03, n=22). This suggests that gain of

function may require additional residues outside of the three identified regions, which will be

subject to further investigation. As an additional approach, we created two point mutations in

which residues 111 and 153 of Gβ1 were replaced with corresponding Gβ5. These residues are

located within the 110-112 and 140-168 stretches were chosen based on the surface exposure on

the Gβ1 crystal structure (see Fig. 5B below). Substitution of Tyr-111 with alanine resulted in a

dramatic reduction of the degree of prepulse relief (I+PP/I-PP) to 1.48±0.19 (n=15), and

replacement of Asp-153 to asparagines resulted in an even stronger reduction of prepulse relief

to 1.37±0.08 (n=15). These data suggest that even single amino acid substitutions in at least two

of the identified regions are sufficient to drastically attenuate the ability of Gβ1 to inhibit N-type

calcium channels, consistent with the idea that the inability of the chimeras to regulate channel

activity did not arise secondarily from global structural changes.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


To confirm the functionality of those Gβ chimeras that were unable to regulate N-type

channel activity, we carried out a series of experiments with G protein coupled inward rectifier

potassium (GIRK) channels. GIRK1 and GIRK4 subunits were coexpressed in tsA-201 cells

with either wild type or chimeric/mutant Gβ subunits, and the whole cell GIRK conductance was

determined via whole call patch clamp recordings using a voltage ramp protocol. Rather than

examining the entire set of chimeras, we focused on the key chimeras and mutants which most

narrowly defined the regions involved in N-type channel modulation, i.e., Gβ1(100-112),

Gβ1(140-168), Gβ1(186-204), Gβ1(Y111A), Gβ1(D153N), and the Gβ5(110-112, 140-168, 186-

204) chimera. As shown in Fig. 4, expression of GIRK1/4 in the absence of exogenous Gβ

subunits resulted in some background GIRK activity, consistent with previous work in Xenopus

oocytes (22). Cotransfection of Gβ1 subunits resulted in a significant increase in whole cell

conductance by ~300%. Interestingly and in contrast with a previous study (22), wild type Gβ5

subunits also effectively activated GIRK1/4 channels, thus confirming that Gβ5 subunits are

indeed functionally expressed in our system. But more importantly, every single one of the

chimeric and mutant Gβ subunits examined mediated a significant increase in GIRK1/4 activity

to a level comparable to that seen with wild type Gβ1. Hence, we conclude that the lack of

effects of these chimeras/mutants on N-type channel activity did not arise from inadequate

protein expression or protein misfolding.

G protein PKC crosstalk

We have shown previously that PKC dependent phosphorylation of Thr-422 in the Cav2.2

calcium channel I-II linker selectively antagonizes the Gβ1 mediated inhibition of N-type channel

activity (18). We also showed that all aspects of this effect could be mimicked by replacing Thr-


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


422 with glutamic acid (17,18), thus eliminating the need for diffuse activation of PKC and

consequently the potential for secondary effects/incomplete specificity of PKC activators. Since

a chimera containing the first 47 residues of Gβ1 (Fig. 5A) appeared to behave like wild type Gβ1

with regard to N-type channel regulation (recall Fig. 1), we examined whether the PKC crosstalk

could still be observed with this construct. While the T422E mutation reduced the ability of Gβ1

to inhibit N-type channel activity, the 5111 chimera failed to recognize the presence of the

T422E substitution (Fig. 5B), thus indicating that a key Gβ1 structural determinant is located in

the N-terminal region. Since the PKC crosstalk is only observed with Gβ1, we examined the N-

terminal 47 amino acid stretch for residues unique to this subunit (see Fig. 5C, bolded letters).

As shown in Fig. 5D, replacement of Asp-5 with glutamic acid had no effect on crosstalk. In

contrast, replacement of Asn-35 and Asn-36 with corresponding sequence in Gβ3 abolished the

effects of the T422E I-II linker mutation, suggesting that one or both of these asparagine residues

sense the presence of a negative charge within the I-II linker G protein binding domain (7). If

so, then one should be able to confer crosstalk onto other G protein subunits by substituting

asparagine residues in complimentary positions. To test this hypothesis, we carried out site

directed mutagenesis in the Gβ3 subunit (which modulates N-type channels effectively, but

whose action is not antagonized by PKC; 18), and compared the abilities of the mutant

Gβ3(S35N, G36N) construct to inhibit wild type and T422E mutant N-type channels. Consistent

with our hypothesis, the mutant Gβ3 subunit inhibited the T422E channel significantly (p<0.05)

less effectively than the wild type Cav2.2 channel (Fig. 5E).

To confirm that the observations were not an artefact of the T422E calcium channel

mutant, we repeated the experiments shown in Figs. 5D and E using proper activation of PKC

via 30 nM of the phorbol ester PMA. For each cell, the degree of prepulse relief was measured


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


before application of PMA and 3 minutes after PMA application. As seen in Fig. 5F, activation

of PKC did not affect the degree of prepulse relief observed with the Gβ1(N35S, N36G) mutant.

In contrast, mutagenesis of Gβ3 residues 35 and 36 to corresponding Gβ1 sequence (i.e.,

Gβ3(S35N, G36N)) conferred the crosstalk behaviour, such that the degree of prepulse was

significantly reduced (p<0.01, paired t-test) following the application of PMA.

Taken together, these data suggest that the Gβ1 subunit contains a precise locus of two

amino acids that allows this subunit to sense the presence of a negative charge (such as a

phosphate group) on the N-type calcium channel α1 subunit.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


Discussion

Comparison with previous work

Our work constitutes the first systematic approach towards delineating key Gβ subunit regions

that are essential for inhibiting N-type calcium channel activity. Unlike previous studies that

were based on alanine mutagenesis of Gβ subunit residues known to be involved in Gα subunit

binding (i.e., 19,20), we employed a chimeric approach that was based on the differential

abilities of Gβ1 and Gβ5 to inhibit N-type calcium channels. We show that mutagenesis of

residues Asn-110, Tyr-111, Val-112, a motif that is highly conserved in all Gβ subunits other

than Gβ5, virtually abolished all G protein regulation of the N-type channel. This region is

located outside of the known Gα interaction domain (23,24; see Fig. 6) and has not been

previously identified as an important functional domain on the Gβ1 subunit. G protein inhibition

was also lost upon substitution of Gβ1 residues 140-168 and 186-204. Hence, the G protein β

subunit structural determinants that control signalling to N-type calcium channels appear to be

different from those implicated in coupling to other effector systems such as adenylyl cyclase,

GIRK channels, or phospholipase β2 (i.e., residues 72-105, residues 115-135, residues 143, 186,

228 and 332 – (19,22, 25-31)). However, in lieu of specific examination of the action of our

chimeras on effectors other than GIRK channels, it is difficult to gage whether the regions

identified in our study are exclusively involved in signalling to N-type channels.

Within regions 140-168 and 186-204 respectively, twelve and nine amino acid residues

are identical in Gβ1 and Gβ5, and can therefore not account for the observed effects (the

conserved residues include Thr-143 and Asp-186 which were implicated previously as being

important for N-type channel modulation; (19)). Examination of the localization of the non-

conserved residues in regions 110-112, 140-168 and 186-204 reveals a pattern in which all of the


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


exposed residues appear to be arranged in a ribbon like fashion at the protein surface opposite to

that containing the Gα interaction domain (see Fig. 6B). Among these exposed residues, Tyr-111

and Asp-153 were individually found to be critical for G protein inhibition. The lack of overlap

with residues known to be involved in Gα binding contrasts with what is observed with a number

of other Gβγ effectors that interact with the Gα interaction region following dissociation of the

heterotrimeric G protein complex (19). It is important to note that our data do not necessarily

disagree with previous findings that mutagenesis of conserved residues within the Gα binding

region can affect Gβ1 inhibition of N-type calcium channels (19,31), as our chimeric approach

was designed to elucidate the molecular basis of the differences between Gβ1 and Gβ5 regulation

of N-type channels, rather than identification of all residues that contribute to N-type channels

inhibition. The notion that our intended gain of function chimera was not sufficient to mediate

significant G protein inhibition of the channel is also consistent with the existence of additional

structural determinants.

Substitution of residues 45-75 of Gβ1 with Gβ5 sequence resulted in a curious

enhancement of the degree of N-type channels inhibition. This stretch of amino acids contains a

leucine residue in position 55, which when mutated to alanine, has been reported to increase N-

type channel inhibition (19), in agreement with present data. This region also contains Ser-67

which, when mutated to the corresponding Gβ5 lysine residue, has been reported to abolish G

protein inhibition altogether (31). Our observation that the 45-75 chimera mediated robust G

protein inhibition appears at odds with this finding, however, it is conceivable that the effects of

the Ser-67 substitution are masked by the enhancing effect of Leu-55. Additional mutagenesis of

individual residues will be required to elucidate the precise roles of residues 55 and 67 in

calcium channel inhibition.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


Taken together, our data indicate that a ribbon like structure comprised of residue Tyr-

111 and residues in regions 140-168 and 186-204 of Gβ1 is essential for N-type channel

inhibition, with residues 111 and 153 being of particular importance.

Molecular Mechanism of G protein – PKC crosstalk

The N-type calcium channel contains at least three separate physical binding sites for Gβγ - two

of these sites are formed by about ~20 amino acid stretches within the domain I-II linker (6,7)

plus an additional site in the C-terminal region (9). The observation that multiple regions within

the Gβγ subunit were found to be critical for G protein inhibition is consistent with the existence

of multiple G protein micro binding sites on the N-type channel α1 subunit. The second of the

two I-II linker Gβγ regions contains the PKC phosphorylation site formed by residue Thr-422

(7,17). In vitro phosphorylation of this site blocks Gβγ binding to this region (7), and

replacement of Thr-422 with glutamic acid mimics all aspects of phosphorylation (17,18). Thus,

the T422E mutation provides a convenient means of investigating G protein β subunit

determinants that contribute to the crosstalk phenomenon, without having to resort to activation

of PKC via pharmacological means.

Our findings indicate that residues Asn-35 and/or Asn-36 near the N-terminal helix of the

Gβ1 subunit were necessary and sufficient for sensing the presence of this glutamic acid residue,

or the true phosphorylation of the native threonine residue. The location of this

“phosphorylation sensor” on the Gβ1 subunit is somewhat surprising, since this region is not

typically associated with coupling to any of the known Gβγ effectors (see above). Yet, the role of

these two residues in supporting G protein–PKC crosstalk appears to be highly specific, as

substitution of these residues into the Gβ3 subunit that is normally incapable of recognizing


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


phosphorylation of Thr-422 (18) conferred the crosstalk behaviour. We can, at this point, only

speculate about the actual mechanism by which Gβ residues 35 and 36 functionally couple to the

N-type channel I-II linker containing the Thr-422 site. We have shown previously that

disruption of Gβγ binding to this region results in a complete loss of G protein inhibition (7).

Hence, if there is a direct interaction between Gβ subunit residues Asn-35/Asn-36 and Thr-422,

the phosphorylation event would only serve to partially disrupt Gβγ binding since G protein

inhibition is merely reduced, but not eliminated following activation of PKC (17,18). On the

other hand, the data obtained with the 5111 chimera together with the low (~30%) degree of

sequence conservation in residues 1-47 suggests that this region of Gβ is not a major determinant

of G protein action on N-type channels. Yet, between residues 44 and 53, the amino acid

sequence of among all Gβ subunits is highly conserved (see Fig. 5C). This raises the possibility

that this region could interact with the N-type channel I-II linker, but that phosphorylation of

residues Thr-422 might weaken binding via an interaction with Gβ1 residues Asn-35 and Asn-36,

thus accounting for the reduced G protein inhibition following activation of PKC. Further

mutagenesis of residues 44-53 will, however, be required to test this hypothesis.

Assuming that a site near/at residues 35 and 36 does indeed interact with the calcium

channel I-II linker region flanking Thr-422, then this begs the question as to which region of the

N-type channel interacts with the ribbon like structure depicted in Fig. 6B (i.e., residues 111,

140-168, 186-204). As stated above, the N-type channel contains two additional binding

domains for Gβγ, a region in the C-terminal (9) and a second I-II linker site ~30 residues

upstream of Thr-422 (6,7). Disruption of the Gβγ binding to this region completely eliminates G

protein inhibition (7), whereas deletion of the carboxy terminus site has only a minor effect (17).

Considering that replacement of these Gβ1 subunit domains with Gβ5 sequence completely


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


abolished G protein action, we thus favour the I-II linker site over the C-terminus as a potential

interacting partner. We therefore envision a model in which Gβγ is held in place through

interactions of two distinct sites on the Gβ protein with two spatially separate regions within the

calcium channel I-II linker. In this model, the functional interaction between the phosphorylated

Thr-422 I-II linker residue and residues 35/36 on the Gβ1 subunit would destabilize the overall

binding interaction, thus reducing the extent of G protein inhibition of the channel. The C-

terminus of the N-type channel might contribute towards stabilizing overall G protein binding

(perhaps by interacting with previously identified residues in the Gα interaction region).

Taken together, our data closes a major gap in our understanding of the molecular basis

underlying crosstalk between G protein and PKC regulation of N-type calcium channels. The

presence of a specific site on the Gβ subunit that serves as a molecular detector of PKC

dependent phosphorylation of the N-type calcium channel provides a unique means of

integrating multiple signalling pathways at the level of a protein-protein interaction. This may

allow for precise regulation of N-type calcium channel activity, and consequently, synaptic

transmission.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


Acknowledgments:

We would like to thank Dr. Terry Snutch for providing wild type calcium channel cDNAs, and

Dr. Hubert van Tol for providing GIRK subunits. This work was supported by an operating

grant to GWZ from the Canadian Institutes of Health Research (CIHR). GWZ is a CIHR

Investigator and holds a senior scholar award from the Alberta Heritage Foundation for Medical

Research (AHFMR). CJD holds a studentship award from the AHFMR, ZPF was supported by

postdoctoral fellowships from the CIHR and the Heart and Stroke Foundation of Canada (HSF).

MIA was supported by a HSF postdoctoral fellowship. Last but not least, we would like to thank

Dr. Scott Jarvis for help with artwork.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


References

1. Wheeler, G.B., Randall, A., & Tsien, R.W. (1994) Science 264, 107-111. 2. Dunlap, K., Luebke, J.I., & Turnerm T.J. (1995) Trends Neurosci. 18, 89-98. 3. Zamponi, G.W. (2001) Cell Biochem Biophys. 34, 79-94. 4. Ikeda, S.R. (1996) Nature 380, 255-258. 5. Herlitze S., Garcia D.E., Mackie K., Hille B., Scheuer T., & Catterall W.A. (1996)

Nature 280, 258-262. 6. DeWaard M, Liu H, Walker D, Scott VE, Gurnett CA, & Campbell KP. (1997) Nature

385, 446-450. 7. Zamponi, G.W., Bourinet, E., Nelson, D., Nargeot, J., & Snutch, T.P. (1997) Nature 385,

242-246. 8. Page, K.M., Stephens, G.J., Berrow, N.S., & Dolphin, A.C. (1997) J. Neurosci. 17, 1330-

1338. 9. Qin, N., Platano, D., Olcese, R., Stefani, E., & Birnbaumer, L. (1997) Proc. Natl. Acad.

Sci. USA. 94, 8866-8871. 10. Garcia, D.E., Li B., Garcia-Ferreiro R.E., Hernandez-Ochoa E.O., Yan K., Gautam N.,

Catterall W.A., Mackie K., & Hille B. (1998) J. Neurosci. 18, 9163-9170. 11. Ruiz-Velasco, V., & Ikeda, S.R. (2000) J. Neuroscience 20, 2183-2191. 12. Arnot, M.I., Stotz, S.C., Jarvis, S.E., & Zamponi, G.W. (2000) J. Physiol. 527, 203-212. 13. Stea, A., Soong, T.W., & Snutch, T.P. (1995) Neuron 15, 929-940. 14. Barrett, C.F., & Rittenhouse, A.R. (2000) J. Gen. Physiol. 115, 277-286. 15. Swartz, K.J. (1993) Neuron 11, 305-320. 16. Swartz, K.J., Merrit, A., Bean, B.P., & Lovinger, D.M. (1993) Nature 361, 165-168. 17. Hamid, J., Nelson D., Spaetgens R., Dubel S.J., Snutch T.P., & Zamponi G.W. (1999) J.

Biol. Chem. 278, 6195-6202. 18. Cooper, C.B., Arnot M.I., Feng Z.P., Jarvis S.E., Hamid J., & Zamponi G.W. (2000) J

Biol Chem. 275, 40777-40781.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


19. Ford, C.E., Skiba N.P., Bae H., Daaka Y., Reuveny E., Shekter L.R., Rosal R., Weng G., Yang C.S., Iyengar R., Miller R.J., Jan L.Y., Lefkowitz R.J., & Hamm H.E. (1998) Science 280, 1271-1274.

20. Agler, H.L., Evans, J., Colecraft, H.M., & Yue, D.T. (2003) J. Gen. Physiol. 121, 495-

510. 21. Feng, Z.P., Arnot, M.I., Doering, C.J., & Zamponi, G.W. (2001) J. Biol. Chem. 276,

45051-45058. 22. Mirshahi, T., Robillard, L., Zhang, H., Hebert, T.E., & Logothetis, D.E. (2002) J. Biol.

Chem. 277, 7348-7355. 23. Wall, M.A., Coleman D.E., Lee E., Iniguez-Lluhi J.A., Posner B.A., Gilman A.G., &

Sprang S.R. (1995) Cell 83, 1047-1058. 24. Lambright, D.G., Sondek J., Bohm A., Skiba N.P., Hamm H.E., & Sigler P.B. (1996)

Nature 379, 311-319. 25. Yan, K. & Gautam, N. (1997) J. Biol. Chem. 272, 2056-2059. 26. Yan, K. & Gautam, N. (1996) J. Biol. Chem. 271, 17597-15600. 27. Chen, Y., Weng G., Li J., Harry A., Pieroni J., Dingus J., Hildebrandt J.D., Guarnieri F.,

Weinstein H., & Iyengar R. (1997) Proc. Natl. Acad. Sci. U S A. 94, 2711-2714. 28. Panchenko, M.P., Saxena K., Li Y., Charnecki S., Sternweis P.M., Smith T.F., Gilman

A.G., Kozasa T., & Neer E.J. (1998) J. Biol. Chem. 273, 28298-28304. 29. Buck, E., Li J., Chen Y., Weng G., Scarlata S., & Iyengar R. (1999) Science 283, 1332-

1335. 30. Albsoul-Younes A.M., Sternweis P.M., Zhao P., Nakata H., Nakajima S., Nakajima Y.,

& Kozasa T. (2001) J. Biol. Chem. 276, 12712-12717. 31. Mirshaji, T., Mittal., V., Zhang, H., Linder, M.E., & Logothetis, D.E. (2002) J. Biol.

Chem. 277, 36345-36350. 32. Sondek, J., Bohm, A., Lambright, D.G., Hamm, H.E., & Sigler, P.B. (1996) Nature 379,

369-374.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


Figure legends:

Figure 1 Effect of chimeric G protein subunits on N-type calcium channel activity. A.

Schematic representation of a chimeric G protein β subunit adapted from the crystal structure

reported by Sondek et al. (31). The Gβ subunit is comprised of an N-terminal helix linked to a

rigid seven bladed propeller structure. An initial set of chimeras was constructed by swapping

fragments between Gβ1 (indicated in blue) and Gβ5 (indicated in orange). For this purpose, the Gβ

sequence was divided into four segments (i.e., N-terminus - residue 47, residues 47- 168,

residues 168-280, and residue 280 - C-terminus; the numbering corresponds to Gβ1 sequence, see

arrows for approximate positions within the Gβ subunit structure). The nomenclature of the

chimeric constructs is based on the origin of the four segments, with the depicted 5151 construct

indicating that segments 1 and 3 were derived from Gβ5. B. Current recordings obtained from N-

type (Cav2.2 + β1b +α2-δ1) calcium channels in the presence of wild type Gβ1, or chimeric Gβ

subunits, before and after application of a strong depolarizing prepulse as outlined in the

Materials and Methods section. In each case, Gγ2 and an EGFP marker were coexpressed. Note

that each of the chimeric constructs results in robust G protein inhibition. C. Summary of the

effects of wild type and chimeric Gβ subunits on N-type channel activity, in the form of ratios of

current amplitudes obtained after and before application of the prepulse. Error bars denote

standard errors, numbers in parentheses reflect numbers of experiments, asterisks indicate

statistical significance (p<0.05) relative to Gβ5 (ANOVA). The two arrows in the bar chart

highlight chimeras (i.e., 1511, 1151) in which substitution of a single region abolished the

ability of Gβ1 to regulate N-type channel activity. The dotted line indicates the level of

modulation seen with Gβ5. Inset: Membrane localization of EGFP-tagged wild type and


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


chimeric G protein β subunits, visualized via bight field light microscopy (top row) or

fluorescence confocal microscopy (bottom row). Note that the cells appear rounded due to our

particular incubation protocol (i.e., 28°C, see Materials and Methods).

Figure 2: Chimeric analysis of residues 47-168 in the G protein β1 subunit. Top: Chimeric G

protein subunits are depicted using the same color schemes as described in Fig. 1. The

numbering on the chimeric structures reflect amino acids in the Gβ1 sequence that were replaced

by corresponding Gβ5 residues, the “plus” and “minus” signs reflect ability or inability to inhibit

N-type channels. Bottom: Bar graph illustrating the degree of prepulse relief obtained upon

coexpression of the N-type calcium channels with the chimeras depicted in the upper part of the

figure. Note the axis break on the ordinate (i.e., the 47-75 construct resulted in dramatically

greater prepulse relief compared to wild type Gβ1). Asterisks indicate statistical significance

(p<0.05, ANOVA) relative to wild type Gβ5. The dotted line indicates the level of modulation

seen with the 1511 construct. All error bars denote standard errors.

Figure 3: Chimeric analysis of residues 168-280 in the G protein β1 subunit. A. Current records

obtained from tsA-201 cells expressing the N-type calcium channel, Gγ2 and G protein β subunit

chimeras 168-204 (top), 204-248 (middle) and 248-280 (top). The experimental conditions were

as outlined in Fig. 1B. Note however, that the duration of the test depolarization was longer in

the records shown here. B. Bar graph illustrating the degree of prepulse relief obtained upon

coexpression of the N-type calcium channels with the chimeras depicted in panel C. Asterisks

indicate statistical significance (p<0.05, ANOVA) relative to wild type Gβ5. The dotted line


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


indicates the level of modulation seen with the 1151 construct. C. Chimeric G protein subunits

are depicted and labeled as described in Figs. 1 and 2. All error bars denote standard errors.

Figure 4 Activation of GIRK1/4 channels by wild type, and selected chimeric and mutant

G protein β subunits. A. Raw current traces elicited by stepping from a holding potential of –35

mV to various test potentials ranging from –130 mV to +60 mV. B. Representative GIRK1/4

currents in the absence or the presence of G protein β subunits, acquired via a ramp protocol as

outlined in the Materials and Methods section. C. Summary of GIRK1/4 activity induced by

wild type and chimeric G protein β subunits in comparison with the activity observed in the

absence of Gβ. Bars indicate mean whole cell GIRK conductance determined via ramp protocols

as described in the Materials and Methods section. Error bars denote standard errors, numbers in

parentheses reflect numbers of experiments, asterisks denote statistical significance relative to -

Gβ.

Figure 5 A. Schematic representation of the 5111 chimera. B. Degree of prepulse relief

observed with wild type Cav2.2 or mutant T422E Cav2.2 calcium channels in the presence of Gβ1

or the 5111 chimera. Note that the replacement of Thr-422 with glutamic acid antagonizes Gβ1

action, but has no effect on modulation by the 5111 chimera. C. Sequence alignment of the N-

terminal helix regions of Gβ1 through Gβ5. Residues shown in bold are unique to Gβ1. D. Effect

of mutagenesis of unique Gβ1 residues in PKC-G protein crosstalk. Note that the antagonistic

effect of the T422E mutation on G protein inhibition is abolished upon mutagenesis of

asparagine residues 35 and 36. E. Induction of G protein – PKC crosstalk into the Gβ3 subunit

after insertion of asparagines residues into positions 35 and 36. All error bars are standard


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


errors, the asterisks denote statistical significance (p<0.05, t-test) between the degree of

inhibition observed with the wild type and T422E mutant N-type channel. F. Effect of protein

kinase C activation on the degree of prepulse relief observed following the coexpression of

Gβ1(N35S,N35G) and Gβ3(S35N, G36N). In each case, the degree of prepulse relief was

measured prior to, and 3 minutes after application of 30 nM PMA. Note that PMA significantly

reduces the effect of Gβ3(S35N,G35N), but not that of the Gβ1 point mutant. Error bars are

standard errors, the asterisk reflects statistical significance at the 0.05 level (paired t-test +PMA

vs. -PMA).

Figure 6 Localization of structural determinants of Gβ1 inhibition of N-type calcium

channels on the three dimensional structure of the Gβ subunit. Protein structure data were

obtained from crystal structure coordinates (32) and visualized via Viewer Pro software

(Accelerys Inc.). A. Residues 35 and 36 are visualized in red and the Asn-110, Tyr-111, Val-

112 motif is indicated in yellow (note that residues 110 and 112 are buried in the protein, with

only Tyr-111 being exposed). Residues shown in blue and green, respectively, reflect amino

acids in regions 140-168 and 186-204. B. Same as panel A, but depicting only those residues

within the identified regions that are not conserved between Gβ1 and Gβ5. Residues depicted in

pink are known to be involved in interactions with the Gα subunit (23,24).


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

10 ms200 pA

Gβ1 Gβ5111

-PP-PP

+PP+PP

-PP

+PP

Gβ5115Gβ1115

-PP

+PP

A

C

BI(+

PP

)/I(-

PP

)

1

2

3

4

( 2 0 ) *

( 9 ) *

(1 1 )

(1 0 )(1 0 ) (1 0 )

( 1 2 ) *

(1 6 ) *

(8 )(2 0 ) (8 ) ( 1 3 )

Gβ1

G β5

5111

5551

5151

5115

1511

1151

5511

1155

1555

1115

( 3 1 )

ctrl

Gβ1

Gβ5

5511

5551

5111

47

168

280

340(COOH)

5151

NH2

by guest on February 10, 2018 http://www.jbc.org/ Downloaded from

http://www.jbc.org/

47-11

6

1511

116-1

68

75-10

0

47-75

NYV-CAI

116-1

4014

0-168

I(+P

P)/I

(-P

P)

1.0

1.5

2.0

2.5

4.5

5.0

(10) (15)

(14)*

(15)*

(15)

(18)*

(16)(25)

-1511116-140

-

140-168 47-116

75-100

47-75

NYV-CAI

116-168

-

+

- +

+

-


http://www.jbc.org/

- 1151

168-204204-248

248-280

168-186 186-204

168-204

204-248

248-280

100 pA

20 ms

-PP

+PP

-PP

-PP

+PP

+PP

A

B

CI(+

PP

)/I(-

PP

)

1

2

(20)

(14)

(15)*

(19)*

(16)*

(11)

1151

168-2

0420

4-248

248-2

8016

8-186

186-2

04

+

+ -

+ -


http://www.jbc.org/

Con

duct

ance

[nS

]

0

2

4

6

8

10

12

14

16

(35)

(40)*(27)*

(35)*(20)* (18)* (17)* (14)* (25)*

-Gβ

Gβ1

Gβ5

NYV-CAI

140-1

6818

6-204

Gβ1(Y

111A

)Gβ

1(D15

3N)

Gβ5(1

10-11

2, 14

0-

168,

186-2

04)

-4 nA

-100 +60

Voltage [mV]

1 nA20 ms

A B

C

Gβ1

-Gβ

140 -168


http://www.jbc.org/

(9)*

(11)

-PMA+PMA

1

2

3

4

5

I(+P

P)/I

(-PP

)

1

2

3

4

5WTT422E

(20)

(10)*

(7)(9)

Gβ1 5111

Gβ1 ........MS ELDQLRQEAE QLKNQIRDAR KACADATLSQ ITNNIDPVGR IQMRTRRTLRGH... Gβ2 ........MS ELEQLRQEAE QLRNQIRDAR KACGDSTLTQ ITDGLDPVGR IQMRTRRTLRGH... Gβ3 ........MG EMEQLKQEAE QLKKQIADAR KACADITLAE LVSGLEVVGR VQMRTRRTLRGH...Gβ4 ........MS ELEQLRQEAE QLRNQIQDAR KACNDATLVQ ITSNMDSVGR IQMRTRRTLRGH... Gβ5 MATDGLHENE TLASLKSEAE SLKGKLEEER AKLHDVELHQ VAERVEALGQ FVMKTRRTLKGH...

AI(+

PP)/I

(-PP)

D

Gβ1(N35S,N36G)Gβ1(D5E)Gβ11

2

3

4

5WTT422E

(20)

(10)*

(6)

(7)*

(18)(10)

C

B

5111

I(+PP

)/I(-P

P)

1

2

3

4 (22)

(11)*

E

WT T422E

FGβ3(S35N,G36N)

Gβ1(N35S,N36G) Gβ3(S35N,G36N)

5

I(+PP

)/I(-P

P)


http://www.jbc.org/

A

B


http://www.jbc.org/

and Gerald W. ZamponiPeloquin, Jawed Hamid, Wendy Barr, Aparna Nirdosh, Brett Simms, Robert J. Winkfein Clinton J. Doering, Alexandra E. Kisilevsky, Zhong-Ping Feng, Michelle I. Arnot, Jean

N-type calcium channels subunit locus controls crosstalk between PKC and G protein regulation ofβA single G

published online April 22, 2004J. Biol. Chem.

10.1074/jbc.M308693200Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/lookup/doi/10.1074/jbc.M308693200

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;M308693200v1&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2004/04/22/jbc.M308693200.citation

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=early/2004/04/22/jbc&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2004/04/22/jbc.M308693200.citation

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/

g protein chimera, 1 a single gβ subunit locus controls crosstalk

Documents