Proteasome involvement in the degradation of the G
q
family of Ga subunits
Bente B. Johansson, Laura Minsaas and Anna M. Aragay
Department of Biomedicine, Faculty of Medicine, University of Bergen, Norway
One common feature of G protein-coupled receptor
(GPCR) signaling is the rapid loss of cellular sensitiv-
ity even in the presence of a stimulus. Insensitivity to
the extracellular stimuli reflects intracellular events
such as receptor⁄ G protein uncoupling, G protein
inactivation, and receptor sequestration and degrada-
tion that together regulate the duration and⁄ or the
magnitude of the signaling event. In particular, the
rapid degradation of signaling proteins by the protea-
some ⁄ ubiquitin system appears to play an important
role in the control of the duration of the signal. For
instance, ligand-stimulated ubiquitination of several
mammalian cell surface receptors has been reported
to induce internalization, followed by degradation in
lysosomes [1]. The ubiquitin-proteasome pathway
influences agonist-induced degradation of opioid
receptors [2], rhodopsin [3] and the yeast pheromone
receptors, ste2p and ste3p [4,5]. Recently, it has been
shown that agonist-stimulated ubiquitination of the b2
adrenergic receptor (b2AR) is required for receptor
degradation, whereas b-arrestin 2 ubiquitination is
essential for rapid receptor internalization [6]. In
addition, the turnover of G protein coupled receptor
kinase 2 (GRK2), the kinase that regulates the dur-
ation of receptor activation, is mediated by the protea-
some [7]. Also, it is becoming increasingly clear that

subunits. Pretreatment
with proteasome inhibitors attenuated the degradation of both G alpha
subunits. In contrast, pretreatment of cells with inhibitors of lysosomal
proteases and nonproteasomal cysteine proteases had very little effect on
the stability of the proteins. Significantly, the turnover of these proteins is
not affected by transient activation of their associated receptors. Fraction-
ation studies showed that the rates of Ga
q
and Ga
16
degradation are accel-
erated in the cytosol. In fact, we show that a mutant Ga
q
which lacks its
palmitoyl modification site, and which is localized almost entirely in the
cytoplasm, has a marked increase in the rate of degradation. Taken
together, these results suggest that the G
q
class proteins are degraded
through the proteasome pathway and that cellular localization and ⁄ or
other protein interactions determine their stability.
Abbreviations
ALLN, N-acetyl-
L-leucyl-L-leucyl-L-norleucinal; GAP, GTPase activating protein; G protein, heterotrimeric guanine nucleotide-binding protein;
GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; PLC, phosphoinositide phospholipase C; PLCb, phosphoinositide
phospholipase C; PMSF, phenylmethylsulphonylfluoride; RGS, regulator of G protein signaling.
FEBS Journal 272 (2005) 5365–5377 ª 2005 FEBS 5365
Activation of GPCRs by specific ligands, promotes
the exchange of GDP for GTP on Ga subunits, result-
ing in the dissociation from the Gbc dimer with the

2+
from intracellular
stores [22]. The G
q
class includes Ga
q
,Ga
11
,Ga
14
and
Ga
15 ⁄ 16
.Ga
q
,Ga
11
and Ga
14
are highly homologous
and have similar activities towards effector activation.
Ga
q
and Ga
11
are ubiquitously expressed. On the con-
trary, Ga
16
expression is confined to hematopoietic
cells derived mainly from early stages of differentiation

class of G
proteins, pulse and chase analysis of metabolically
labeled cells were performed. For this, HEK293 cells
were incubated for 30 min in presence of [
35
S]methio-
nine and then chased in the presence of unlabeled
medium for various time points. Subsequently, cells
were lysed and immunoprecipitated with the Ga
q
-spe-
cific antibodies for the recovery of proteins from the
membrane. The specificity of antibodies was verified
by analyzing HEK293 cells transiently transfected with
Ga
q
and Ga
16
cDNAs and immunoprecipitating with
the anti-Ga
q
(CT-12872 or sc-392) and anti-Ga
16
(CT56) antibodies prior to pulse-chase experiments. As
shown in Fig. 1(A,B), both anti-Ga
q
Igs detected a
band of 42 kDa that was more prominent in HEK293
cells transient transfected with the plasmid encoding
for Ga

anti-Ga
16
CT56, followed by SDS ⁄ PAGE (A
and C, 12.5% and B, 10% PAGE). The
figure shows representative autoradio-
graphies of whole SDS ⁄ PAGE gels loaded
with the
35
S immunoprecipitates and the
arrowheads indicate the position of Ga
q
and
Ga
16
. The molecular mass standards are
indicated. The arrow indicates the position
of some unspecific bands.
Proteasome degradation of G
q
proteins B. B. Johansson et al.
5366 FEBS Journal 272 (2005) 5365–5377 ª 2005 FEBS
the Ga
16
protein (Fig. 1C). Nevertheless, some unspe-
cific bands of higher molecular mass appear and are
labeled with arrows.
Figure 2A shows a representative experiment of the
pulse chase analysis of endogenous Ga
q
. As can be

are shown in Fig. 2B,C (293). Taken
together, these results suggest that the Ga
q
and Ga
16
proteins either endogenous or overexpressed display a
rapid turnover in HEK293 cells.
To investigate which proteases were responsible for
the degradation of Ga subunits, assays were performed
using cell-permeable protease inhibitors. Pulse and
chase experiments were performed after 3 h of preincu-
bation in the presence of the specific protease inhibitors
of different proteolytic pathways (Fig. 3). Leupeptin
(100 lgÆmL
)1
), an inhibitor of protein degradation in
lysosomes, had no effect on the stability of the Ga
q
and Ga
16
proteins. The presence of N-acetyl-l-leucyl-
l-leucyl-l-norleucinal (ALLN; 1 lm), which blocks non-
proteasomal proteases at 1 lm doses, did not influence
A
B
C
Fig. 2. Ga
q
and Ga
16

16
CT56) followed by SDS ⁄ PAGE
(A, 10%; B and C, 12.5% PAGE). Control immunoprecipitates of
cells expressing only endogenous Ga
q
(293) or in absence of Ga
16
(293) are shown in (B) and (C). The relative amounts of [
35
S]Ga
q
and [
35
S]Ga
16
were determined using a phophoimager and plotted
as a function of the chase time. Single experiments were per-
formed with triplicate samples and the mean of triplicates was nor-
malized by the mean at time zero. Data represent the mean of at
least four independent experiments where error bars are standard
deviations. Upper unspecific are shown by arrows. There are smalls
variations in the amount of these bands but no correspondence in
seen with the decrease G protein content during the chase taking
into account all experiments performed. Arrowheads indicate the
position of Ga
q
and Ga
16
.
B. B. Johansson et al. Proteasome degradation of G

35
S]methionine labeling. Cells were metabolically labeled, chased for the indicated hours and protein extracts
(900 lg from endogenous G
q
expressing cells and 100 lg from transfected cells) were immunoprecipitated and analyzed by SDS ⁄ PAGE.
(A) Representative autoradiographies of endogenous Ga
q
(293), transfected Ga
q
(293 + Ga
q
) or transfected Ga
16
(293 + Ga
16
). The relative
amounts of the [
35
S]Ga subunits were determined using a phosphoimager and plotted as a function of treatment: (B) transfected Ga
q
;
(C) endogenous Ga
q
; and (D) transfected Ga
16
. Data represent mean of triplicates from a single experiment normalized by the mean at zero
hours where error bars are standard deviations. At least three independent experiments obtained similar results. (E) HEK293 cells transiently
transfected with either M2 muscarinic receptor or control vector in presence or absence of Ga
16
or with M1 muscarinic receptor and Ga

markedly increased after 3 h of preincubation with the
proteasome inhibitor MG132. Similar observations
were made in cells transiently transfected with the
muscarinic receptor 2 (M2R) and Ga
16
.
To explore whether receptor activation can modulate
Ga-turnover, receptors for carbachol (M1R and M2R)
were transfected in HEK293 cells that do not express
these receptors endogenously. These receptors were
chosen as it is well established the specificity of coup-
ling of G
q
with M1 receptor and G
16
with M2 recep-
tor. Cells were transfected with pCISM1R and analysis
of endogenous Ga
q
half-life after carbachol activation
was performed by pulse-chase (Fig. 4A). Under control
AB
CD
Fig. 4. Activation of Ga
q
and Ga
16
does not alter the half-life of the protein. HEK293 cells were transiently transfected with plasmids encoding
for M1R (A) and Ga
q

35
S]Ga
16
were determined using a phophoimager and plotted
as a function of the chase time. Data represent the mean of triplicates from a single representative experiment normalized by the mean at
zero hours where error bars are standard deviations. Representative autoradiographies are shown and arrow heads indicate the positions of
Ga
q
and Ga
16
. A minimum of three independent experiments obtained similar results in the all experiments shown.
B. B. Johansson et al. Proteasome degradation of G
q
proteins
FEBS Journal 272 (2005) 5365–5377 ª 2005 FEBS 5369
conditions, the levels of Ga
q
decay were essentially as
described in Fig. 2(A,B). Agonist stimulation did not
alter the degradation rate with 60 and 40% of the pro-
tein remaining at 3 and 6 h, respectively, both for the
ligand-activated and nonactivated Ga
q
. Adding new
media containing 10 lm carbachol every 30 min during
the chase period did not have any effect on the rate of
degradation either (data not shown). More surpris-
ingly, the activated mutant of Ga
q
,Ga

16
. As observed in
Fig. 5A, treatment of M1R-expressing cells with car-
bachol induces the typical increase responsiveness on
inositol phosphates. Equivalent results were observed
AB
CD
Fig. 5. Functional assay of the Ga subunits. HEK293 cells were transiently transfected with or without receptor in presence or absence of
Ga subunit and labeled with myo-[2-
3
H]inositol (10 lCiÆmL
)1
) for 24 h prior to treatment with ligand for 25 min. (A) Cells expressing the M1
muscarinic receptor in presence of endogenous Ga
q
and un-treated or treated with carbachol (10 lM). (B) Cells expressing Ga
q
,Ga
q
R183C
or control vector in absence of M1 receptor and treated as in (A). (C) Cells expressing Ga
16
and M2R and treated as in (A). (D) Cells expres-
sing Ga
16
,Ga
16
Q212L or control vector in absence of M2 receptor and treated as in (A). Expression of the G proteins in each of the assays
is shown in the lower panel. Values represent means of duplicate determinants from a single experiment, which is representative of
minimum two such experiments.

previous results that show no difference in the degra-
dation rate between the stimulated and nonstimulated
G proteins in both crude cytoplasmic and particulated
fractions and for both endogenous and overexpressed
Ga
q
and M1R or Ga
16
and M2R (Fig. 6A–C). How-
ever, a somehow surprising result was the fact that the
crude cytoplasmic fractions of both Ga
q
and Ga
16
were less stable than the membrane-enriched fraction.
After 6 h only 20% of the cytosolic proteins were
remaining vs. 50–60% of the membrane fraction.
These results are consistent with the idea that the
Ga subunits are more stable in the membrane than in
the cytoplasm. To study this further we designed a
mutant Ga
q
protein where the two palmitoylated cys-
teine residues CC9 ⁄ 10 were mutated to serine residues.
For Ga
q
and Ga
11
, subcellular distribution and the
role of N-terminal palmitoylation has been extensively

35
S]Ga
q
and [
35
S]Ga
16
were determined using
a phophoimager and were plotted as a function of chase time. The
amount of Ga
q
and Ga
16
at time zero was set to 100%. The data
represent mean of triplicates from a single representative experi-
ment normalized by the mean at time zero where error bars are
standard deviations. A two-tailed Student’s t-test was run to com-
pare membrane fraction and cytosolic fraction. All tests show a sig-
nificance of *, **P < 0.001 where n varies from 5 to 9. No
significant difference was seen between ligand stimulated and
nonstimulated cells in the same experiments. Representative auto-
radiographies are shown and arrowheads indicate the positions of
Ga
q
and Ga
16
. Upper nonspecific bands are shown by arrows.
B. B. Johansson et al. Proteasome degradation of G
q
proteins

could also be explained by changes in translation or
maturation of the protein. An increase was also
observed in the rate of degradation of the remaining
mutant protein (20% left of the protein) vs. the wild
type (40% left of the protein) at 3 h of chase
(Fig. 7A,B). We also analyzed the rate of degradation
of another mutant Ga
q
protein, Ga
q
IE25 ⁄ 26AA. This
mutant protein has two residues substituted to alanine
in the putative Gbc binding site [34,35]. An equivalent
region in Ga
i
was shown before to be in direct contact
to Gbc [36]. As shown in Fig. 7(A,B), no change in
the total amount of protein or in the rate of degrada-
tion was observed, which is an indication that the
binding of Ga to Gbc subunits may not the limiting
factor for its stability.
Discussion
In this report we present evidence that endogenous
Ga
q
, transfected Ga
q
and Ga
16
proteins degrade with

GRK2 degradation through proteasome is enhanced
by GPCR stimulation [7]. Chronic exposure to ligand
produces a decrease in G protein levels [21]. On the
other hand, our results have provided evidence for the
lack of ligand-induced degradation of Ga
q
proteins.
Neither receptor-activation of Ga
q
or Ga
16
in total
lysates nor in membrane or cytoplasm fractions had
any effect on the half-life of the proteins. Muta-
tional activation of Ga
q
through the inhibition of its
GTPase-activity, did not produce any enhancement in
the rate of degradation compared with the wild-type
protein. Our data is more consistent with an increased
destabilization of the protein in the cytoplasm, a pro-
cess that, in the case of the G
q
family of proteins, is
independent of receptor activation. Short-term receptor
A
B
Fig. 7. The mutant Ga
q
CC9 ⁄ 10SS has an increased rate of degrada-

q
proteins does not induce translocation
of these subunits to the cytoplasm [31]. On the con-
trary, ligand activation of G
s
proteins induces trans-
location of these G proteins to the cytoplasm [40,41],
which could explain previous results showing that lig-
and activation of receptors coupled to G
s
promotes an
increase in the degradation of this subunit [41]. On the
other hand, chronic agonist treatment of receptors
coupled to Ga
q
could deplete the cytoplasmic mem-
brane from receptors and proteins associated to them,
and could explain the increased degradation of Ga
q
in
the experiments with persistent ligand stimulation [42–
44]. Interestingly, very recent work done with the yeast
Ga subunit Gpa1 have shown that poly ubiquitinated
Gpa1 exhibits a cytoplasmic localization [45]. On the
contrary a Gpa1 mutant that lacks the ubiquitinated
subdomain remains unmodified and is predominantly
localized at the plasma membrane.
Protein stability in the plasma membrane can be a
consequence of receptor association, but interactions of
Ga

Recent results have described an RGS–GAIP-inter-
acting protein, GIPN, that has E3-ubiquitin ligase
activity and promotes proteasome-dependent degrada-
tion of Ga
i3
[46]. The role of these proteins in the turn-
over of the Ga
q
protein should be further investigated.
Interestingly, G
q
, apparently without Gbc subunits,
stably associates with caveolin in caveolae structures
[47]. Caveolin has been suggested to act as a scaffold to
trap and stabilize Gq. On the other hand, the degrada-
tion of Ga
o
via the proteasome pathway is protected
by interaction of the Ga
o
subunit with Hsp90 [39]. Also
the Ga
12
subunit, which localizes in membrane frac-
tions [48], has been shown to associate to Hsp90 and
its association is important for Ga
12
signaling [49].
Work in progress indicates that the same interaction
could be taking place for Ga

were obtained from Santa Cruz Biotechnologies (Santa
Cruz, CA, USA). Secondary HRP-labeled antibody was
ordered from Zymed Laboratories. [
35
S]Methionine was
ordered from Amersham Pharmacia Biotech (Piscataway,
NJ, USA). Myo-[2-
3
H]inositol was purchased from Ameri-
can Radiolabeled Chemicals Inc (Saint Louis, MO, USA).
Enhanced chemiluminescence reagents were obtained from
Amersham Pharmacia Biotech. All other reagents were of
the highest grade commercially available.
DNA constructs
The cDNAs from Ga
q
and Ga
16
cloned into pCIS were
provided by M.I. Simon (California Institute of Technol-
ogy). The mutant deficient of Gbc-binding was generated
by using site-directed mutagenesis using pCISGa
q
as a tem-
plate and the following oligos: Ga
q
IE25 ⁄ 26AA: 5¢-ggat
caacgacgaggccgcgcggcagctgcgcaggg-3¢,Ga
q
IE25 ⁄ 26AA-cccc

Cell culture and transfection
HEK293 cells were cultured in Dulbecco’s modified Eagle’s
medium, supplemented with 10% (v ⁄ v) fetal bovine serum
at 37 °C in a humidified atmosphere containing 5% (v ⁄ v)
CO
2
. Transient transfections were performed on 70–80%
confluent monolayers by using LipofectAMINE reagent
according to the manufacturer’s instructions. Briefly,
HEK293 cells (1.5 · 10
6
cells) were seeded on a P60-plate a
day prior to transfection with 5 lg of total plasmid DNA:
pCISGa
q
or pCISGa
16
in presence or absence of pCISM1R
or pCISM2R ⁄ pCISC5aR (Ga ⁄ R ¼ 0.4 ⁄ 0.6), respectively.
The total amount of DNA was kept constant with the addi-
tion of pCISLacZ.
Metabolic labeling
Metabolic labeling was performed 48 h following transfec-
tion of cells kept with Dulbecco’s modified Eagle’s medium,
supplemented with 10% (v ⁄ v) fetal bovine serum. Cells
expressing Ga
q
or Ga
16
were incubated for 1 h in methion-

[50 mm Tris pH 7.5, 300 mm NaCl, 1% (w ⁄ v) n-dodecyl-
b-d-maltoside, 0.1% (w ⁄ v) sodium dodecyl sulfate and 0.5%
(w ⁄ v) deoxycholate, with protease inhibitors] for 1 h at 4 °C
with continuous rocking. In early experiments, the total pro-
tein content in the samples was estimated before immuno-
precipitation by using Bradford analysis, later this step was
omitted due to good reproducibility of the samples. Protein
extracts (900 lg of total protein for endogenous Ga
q
samples and 100 lg for Ga
q
and Ga
16
transfected cells) sup-
plemented with 1 lgÆlL
)1
BSA were immunoprecipitated
overnight at 4 °C with the specific Ga
q
or Ga
16
antibodies,
followed by incubation with protein A-sepharose beads for
1.5 h. Immune complexes were then washed four times with
NaCl ⁄ P
i
, pH 7.2. Following SDS ⁄ PAGE resolution, the gel
was dried and later analyzed by phosphoimaging in a BAS
5000 system from Fuji (Fuji Foto Film, Tokyo, Japan). The
background level was subtracted from the values registered

) for 10 min. The mixture
was centrifuged and neutralized. After centrifugation, the
supernatants were subjected to anion exchange chromato-
graphy. The final eluant was dissolved in scintillation liquid
and counted in a scintillation counter.
Western blotting
For total G protein content analysis, cell extracts were pre-
pared by lysis in a hypotonic buffer (50 mm Hepes, 0.2 mm
EDTA, 1 mm dithiotreitol, pH 7.4) and cleared by centri-
fugation at 500 g for 5 min. Supernatants were boiled in Lae-
mmli sample buffer and resolved by SDS ⁄ PAGE. Proteins
were transferred to a nitrocellulose membrane and probed
with either Ga
16
or Ga
q
antibodies, respectively. Blots were
developed using a chemiluminiscence assay method.
Subcellular fractionation
Cells (1.5 · 10
6
) were seeded for transfection of cells with
Ga
q
and G a
16
, and 5 · 10
6
cells were used for studying
endogenously expressed Ga

1 Bonifacino JS & Weissman AM (1998) Ubiquitin and
the control of protein fate in the secretory and endo-
cytic pathways. Annu Rev Cell Dev Biol 14, 19–57.
2 Chaturvedi K, Bandari P, Chinen N & Howells RD
(2001) Proteasome involvement in agonist-induced
down-regulation of mu and delta opioid receptors.
J Biol Chem 276, 12345–12355.
3 Obin MS, Jahngen-Hodge J, Nowell T & Taylor A
(1996) Ubiquitinylation and ubiquitin-dependent proteo-
lysis in vertebrate photoreceptors (rod outer segments).
Evidence for ubiquitinylation of Gt and rhodopsin.
J Biol Chem 271, 14473–14484.
4 Roth AF & Davis NG (1996) Ubiquitination of the
yeast a-factor receptor. J Cell Biol 134, 661–674.
5 Hicke L & Riezman H (1996) Ubiquitination of a yeast
plasma membrane receptor signals its ligand-stimulated
endocytosis. Cell 84, 277–287.
6 Shenoy SK, McDonald PH, Kohout TA & Lefkowitz
RJ (2001) Regulation of receptor fate by ubiquitination
of activated beta 2-adrenergic receptor and beta-
arrestin. Science 294, 1307–1313.
7 Penela P, Ruiz-Gomez A, Castano JG & Mayor F Jr
(1998) Degradation of the G protein-coupled receptor
kinase 2 by the proteasome pathway. J Biol Chem 273,
35238–35244.
8 Davydov IV & Varshavsky A (2000) RGS4 is arginy-
lated and degraded by the N-end rule pathway in vitro.
J Biol Chem 275, 22931–22941.
9 Bokkala S & Joseph SK (1997) Angiotensin II-induced
down-regulation of inositol trisphosphate receptors in

17 Chidiac P & Ross EM (1999) Phospholipase C-beta1
directly accelerates GTP hydrolysis by Galphaq and
acceleration is inhibited by Gbeta gamma subunits.
J Biol Chem 274, 19639–19643.
18 Sallese M, Mariggio S, D’Urbano E, Iacovelli L & De
Blasi A (2000) Selective regulation of Gq signaling by
G protein-coupled receptor kinase 2: direct interaction
of kinase N terminus with activated galphaq. Mol
Pharmacol 57, 826–831.
19 Usui H, Nishiyama M, Moroi K, Shibasaki T, Zhou J,
Ishida J, Fukamizu A, Haga T, Sekiya S & Kimura S
(2000) RGS domain in the amino-terminus of G
protein-coupled receptor kinase 2 inhibits Gq-mediated
signaling. Int J Mol Med 5, 335–340.
20 Chen CA & Manning DR (2001) Regulation of G pro-
teins by covalent modification. Oncogene 20, 1643–1652.
21 Bohm SK, Grady EF & Bunnett NW (1997) Regulatory
mechanisms that modulate signalling by G-protein-
coupled receptors. Biochem J 322, 1–18.
22 Simon MI, Strathmann MP & Gautam N (1991) Diver-
sity of G proteins in signal transduction. Science 252,
802–808.
23 Grant KR, Harnett W, Milligan G & Harnett MM
(1997) Differential G-protein expression during B- and
T-cell development. Immunology 90, 564–571.
24 Amatruda TT, 3rd Steele DA, Slepak VZ & Simon MI
(1991) G alpha 16, a G protein alpha subunit specifically
B. B. Johansson et al. Proteasome degradation of G
q
proteins

tion and by elimination of palmitoylation sites, but not
be activation mediated by receptors or AlF4. J Biol
Chem 276, 4227–4235.
32 Wedegaertner PB, Chu DH, Wilson PT, Levis MJ &
Bourne HR (1993) Palmitoylation is required for
signaling functions and membrane attachment of
Gq alpha and Gs alpha. J Biol Chem 268, 25001–
25008.
33 McCallum JF, Wise A, Grassie MA, Magee AI, Guzzi
F, Parenti M & Milligan G (1995) The role of palmi-
toylation of the guanine nucleotide binding protein G11
alpha in defining interaction with the plasma membrane.
Biochem J 310, 1021–1027.
34 Evanko DS, Thiyagarajan MM & Wedegaertner PB
(2000) Interaction with Gbetagamma is required
for membrane targeting and palmitoylation of
Galpha(s) and Galpha(q). J Biol Chem 275, 1327–
1336.
35 Evanko DS, Thiyagarajan MM, Siderovski DP &
Wedegaertner PB (2001) Gbeta gamma isoforms selec-
tively rescue plasma membrane localization and palmi-
toylation of mutant Galphas and Galphaq. J Biol Chem
276, 23945–23953.
36 Lambright DG, Sondek J, Bohm A, Skiba NP, Hamm
HE & Sigler PB (1996) The 2.0 A
˚
crystal structure of a
heterotrimeric G protein. Nature 379, 311–319.
37 Madura K & Varshavsky A (1994) Degradation of G
alpha by the N-end rule pathway. Science 265, 1454–

down-regulation of Gq alpha ⁄ G11 alpha (pertussis
toxin-insensitive) G proteins in alpha T3–1 gonadotroph
cells reflects increased G protein turnover but not altera-
tions in mRNA levels. Proc Natl Acad Sci USA 92,
1886–1890.
45 Wang Y, Marotti LA Jr, Lee MJ & Dohlman HG
(2005) Differential regulation of G protein alpha
subunit trafficking by mono- and polyubiquitination.
J Biol Chem 280, 284–291.
46 Fischer T, De Vries L, Meerloo T & Farquhar MG
(2003) Promotion of G alpha i3 subunit down-regula-
tion by GIPN, a putative E3 ubiquitin ligase that inter-
acts with RGS-GAIP. Proc Natl Acad Sci USA 100,
8270–8275.
47 Oh P & Schnitzer JE (2001) Segregation of hetero-
trimeric G proteins in cell surface microdomains: G(q)
binds caveolin to concentrate in caveolae, whereas G(i)
and G(s) target lipid rafts by default. Mol Biol Cell 12,
685–698.
48 Yamazaki J, Katoh H, Yamaguchi Y & Negishi M
(2005) Two G(12) family G proteins, Galpha(12) and
Galpha(13), show different subcellular localization.
Biochem Biophys Res Commun 332, 782–786.
5376 FEBS Journal 272 (2005) 5365–5377 ª 2005 FEBS
Proteasome degradation of G
q
proteins B. B. Johansson et al.
49 Vaiskunaite R, Kozasa T & Voyno-Yasenetskaya TA
(2001) Interaction between the G alpha subunit of
heterotrimeric G(12) protein and Hsp90 is required for