15 BFBE562E-CA98-4347-9404-16A24D946A0D
Glucose-dependent Insulinotropic Polypeptide (GIP)
Downregula
t
ion of a GPCR by β-Arrestin2-mediated Switch from
an Endosomal to a TGN Recycling Pathway
Nazish Abdullah1, Muheeb Beg1, David Soares1, Jeremy S. Dittman1, and Timothy E
McGraw1,2,*
1Department of Biochemistry, Weill Cornell Medicine, 1300 York Ave, New York, New York 10065
2Department of Cardiothoracic Surgery, Weill Cornell Medicine, 1300 York Ave, New York, New
York 10065
Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone involved in nutrient
homeostasis. GIP receptor (GIPR) is constitutively internalized and returned to the plasma
membrane, atypical behavior for a G protein-coupled receptor (GPCR). GIP promotes GIPR
downregulation from the plasma membrane by inhibiting recycling without affecting
internalization. This transient desensitization is achieved by altered intracellular trafficking of
activated GIPR. GIP stimulation induces a switch in GIPR recycling from a rapid endosomal to a
slow TGN pathway. GPCR kinases and β-arrestin2 are required for this switch in recycling. A
coding sequence variant of GIPR, which has been associated with metabolic alterations, has
altered post-activation trafficking characterized by enhanced downregulation and prolonged
desensitization. Downregulation of the variant requires β-arrestin2 targeting to the TGN but is
independent of GPCR kinases. The single amino acid substitution in the variant biases the receptor
to promote GIP stimulated β-arrestin2 recruitment without receptor phosphorylation, thereby
enhancing downregulation.
*Lead author: Timothy E McGraw- temcgraw@med.cornell.edu.
Author Contributions.
N.A., Planned and conducted experiments, interpreted data, prepared figures and edited manuscript.
M.B., Planned and conducted experiments, interpreted data, prepared figures and commented on manuscript.
D.S., Conducted experiments, interpreted data, prepared figures.
J.D., Performed kinetic modeling, interpreted data, prepared figures and edited manuscript.
T.E.M., Conceived project, planned experiments, interpreted data, prepared figures, wrote manuscript
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Author manuscript
Cell Rep. Author manuscript; available in PMC 2017 December 13.
Published in final edited form as:
Cell Rep. 2016 December 13; 17(11): 2966–2978. doi:10.1016/j.celrep.2016.11.050.
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G protein-coupled receptors (GPCRs) are the largest family of signaling receptors. Signal
transduction is initiated downstream of ligand binding via activation of trimeric G proteins
(Lefkowitz and Shenoy, 2005). Post-activation trafficking of GPCRs contributes
significantly to the biological effects of GPCRs (Hanyaloglu and von Zastrow, 2008). In the
majority of cases GPCR activation leads to enhanced internalization and redistribution of
receptors from the plasma membrane (PM) to the interior of cells (Claing et al., 2002). The
resultant reduced PM receptor density desensitizes cells to further ligand stimulation. The
length of this refractory period, which varies among receptors, is important in sculpting the
cellular response. Some GPCRs are internalized and delivered to lysosomes for degradation,
in which case re-sensitization to further ligand stimulation requires synthesis of new
receptors. Other GPCRs are not degraded but rather are recycled back to the PM. The
intracellular dwell time varies among recycled GPCRs, although typically the time to re-
sensitization is shorter than for those GPCRs that require new synthesis.
GPCR kinases (GRK) and β-arrestin proteins are important in the post-activation behavior
of GPCRs (Claing et al., 2002; Smith and Rajagopal, 2016). GRKs phosphorylate agonist
activated GPCR. β-Arrestins, which bind to phosphorylated GPCRs, impact GPCR behavior
in a number of ways (Lohse and Hoffmann, 2014). First, β-arrestin binding blocks trimeric
G protein re-binding to the GPCR, inhibiting further G protein activation by the GPCR.
Second, β-arrestins serve as adaptors that link GPCRs to membrane protein trafficking
machinery, thereby influencing post-activation trafficking of the GPCR. The best-described
example is β-arrestin-mediated targeting to the clathrin-coated pits to stimulate
internalization and induce desensitization (Goodman et al., 1996). However, β-arrestins can
also influence the intracellular trafficking of GPCRs (Shenoy et al., 2009). The duration of
β-arrestin interaction with the GPCR also affects intracellular trafficking and recycling of
GPCRs and GPCRs can be divided into two classes based on their interaction with β-
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arrestins. (Lohse and Hoffmann, 2014). Typically, GPCRs with prolonged interactions with
β-arrestins undergo sustained downregulation. De-phosphorylation of GPCRs reverses β-
arrestin binding, contributing to re-sensitization and GPCR signaling homeostasis (Krueger
et al., 1997). Finally, the β-arrestins link GPCRs to signaling cascades other than those
mediated by trimeric G proteins, including but not limited to ERK, JNK and AKT signaling
pathways, thereby expanding the signaling repertoire downstream of GPCRs (Lefkowitz et
al., 2006).
Glucose-dependent insulinotropic polypeptide (GIP) and Glucagon-like polypeptide-1
(GLP-1) are secreted by endocrine cells of the gut in response to nutrients in chyme (Dupre
et al., 1973). These peptide hormones, known as the incretins, have prominent roles in the
regulation of whole body metabolism, largely through enhancement of glucose-stimulated
insulin secretion by pancreatic β-cells, although they also have effects on other cells,
including adipocytes, osteoblasts and neurons (McIntosh et al., 2009; Tseng and Zhang,
2000). Both incretins signal through GPCRs. The GIP receptor (GIPR) is coupled to trimeric
Gαs signal transduction. Trafficking of GIPR does not conform to the typical GPCR
behavior (Mohammad et al., 2014). In adipocytes, a cell type that natively expresses GIPR,
the receptor is constitutively internalized and recycled to the PM independent of GIP
stimulation. GIP stimulation causes a downregulation of PM GIPR by inducing a slowing of
receptor recycling without affecting internalization. Thus, unlike the canonical GPCR
behavior, GIP-induced downregulation of GIPR is not achieved through stimulation of
internalization but rather by the slowing of recycling (Mohammad et al., 2014).
A naturally occurring coding sequence variant of the human GIPR has been associated in
genome wide association studies with obesity, cardiovascular disease and an increased risk
of bone fractures (Nitz et al., 2007; Sauber et al., 2010; Saxena et al., 2010). In the variant, a
glutamine replaces a glutamate within the 6th transmembrane domain (position 354, GIPR-
Gln354, rs1800437) of the GIPR. The Gln354 substitution alters post-activation trafficking
of the variant rather than ligand binding or cAMP production. The Gln354 substitution has
no effect on receptor trafficking in unstimulated adipocytes. However, upon GIP stimulation,
there is enhanced receptor desensitization. In addition the refractory period of the variant
receptor is four hours as compared to one hour in the wild type (Mohammad et al., 2014).
These data suggest that the altered post-activation trafficking of the GIPR-Gln354 variant
underlies the link of this variant to alterations in human metabolism, emphasizing the
importance of post-activation trafficking for the biology of the GIPR. GIPR trafficking is not
understood in detail at a molecular level nor is it known why the Gln354 substitution affects
GIPR trafficking.
β-Arrestin2 is responsible for GIP-induced downregulation of GIPR
GIPR trafficking does not conform to the canonical GPCR mechanism of downregulation
based on ligand-stimulated internalization. To determine how this specialization is reflected
in the protein machinery that regulates GIPR trafficking, we investigated the roles of
proteins that regulate trafficking of other GPCRs. β-Arrestins, mediate GPCR internalization
by targeting activated receptors to clathrin-coated pits or other internalization mechanisms
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(Lefkowitz et al., 2006). To characterize the role of β-arrestins in GIPR trafficking, we
investigated the impact of siRNA-mediated knockdown of the β-arrestins on the distribution
of GIPR between the PM and intracellular compartments of basal and GIP-stimulated
adipocytes. We used quantitative fluorescence microscopy and a previously characterized
and validated GIPR reporter, HA-GIPR-GFP (HA epitope fused to the extracellular amino
terminus of GIPR and GFP fused to the cytoplasmic C terminus) to study GIPR trafficking
(Mohammad et al., 2014). GIP binds, activates and induces downregulation of HA-GIPR-
GFP similarly to native GIPR, documenting the HA and GFP modifications do not alter the
function of GIPR. HA-GIPR-GFP in the PM is quantified by anti-HA indirect
immunofluorescence of fixed non-permeabilized adipocytes, and GFP fluorescence is
quantified as a measure of total HA-GIPR-GFP expressed per cell. The ratio of anti-HA to
GFP fluorescence is a quantitative measurement of the fraction of HA-GIPR-GFP in PM of
cells (Lefkowitz et al., 2006).
In unstimulated adipocytes GIPR is nearly equally distributed between the interior and PM
of cells, maintained by constitutive internalization and recycling (Mohammad et al., 2014).
Intracellular GIPR is localized to a peri-nuclear compartment and to vesicles (puncta)
distributed throughout the cytosol (Fig 1A). The stimulation of GIPR with GIP, leads to a
reduction of the GIPR from surface. GPCR kinases (GRKs) and β-arrestins mediate
downregulation of most GPCRs (Lefkowitz et al., 2006). We first determined the
requirement for β-arrestins in GIPR trafficking. Using siRNA for transient knockdown in
adipocytes, we achieved an 85% depletion of nonvisual β-arrestin1 and a 65% depletion of
nonvisual β-arrestin2, as determined by rt-PCR 24 hrs following introduction of the siRNAs
(Supplementary information, Fig S1 and SuppTable 1). Knockdown of β-arrestin1 or 2 did
not grossly alter the overall intracellular localization of GIPR in basal adipocytes (Fig 1A).
GIP stimulation induces a 30% decrease of GIPR in the PM (Mohammad et al., 2014).
Depletion of β-arrestin2 but not the depletion of β-arrestin1 abrogated GIP-stimulated
receptor downregulation (Fig 1B). Knockdown of neither of the β-arrestins affected the
distribution of GIPR between the PM and the interior of unstimulated adipocytes.
Simultaneous knockdown of both β-arrestins did not have an affect beyond knockdown of β-
arrestin2 alone (Fig 1B). GIPR downregulation was restored to β-arrestin2 knockdown
adipocytes by re-expression of β-arrestin2, confirming the effect of the knockdown was due
to a depletion of β-arrestin2 (Fig 1B). These data establish that β-arrestin2 is required for
GIP-induced receptor downregulation. These data also suggest that neither β-arrestin
isoform has a role in GIPR trafficking in unstimulated adipocytes because the distribution of
GIPR between the PM and intracellular compartments in basal adipocytes, which is
maintained by constitutive internalization and recycling, was unaffected by depletion of β-
arrestin isoforms individually or together.
β-Arrestin2 is responsible for GIP-induced slowing of GIPR recycling
GIPR downregulation is achieved by GIP-induced slowing of exocytosis with no effect on
endocytosis (Mohammad et al., 2014). We measured GIPR endocytosis to determine if the
effect of β-arrestin2 depletion on downregulation was indirect due to an effect on
internalization. The HA-GIPR-GFP internalization rate constant was measured by
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quantifying the uptake of anti-HA antibody divided by the amount of HA-GIPR-GFP on the
PM, an assay adapted from studies of trafficking of the Glut4 glucose transporter (Blot and
McGraw, 2006). A plot of the GIPR internal-to-PM ratio yields a straight line whose slope is
the internalization rate constant. Consistent with our previous studies, GIP activation of
GIPR had no effect on the internalization rate constant (Fig 1C & E). β-Arrestin2 depletion
did not affect the GIPR internalization in basal or GIP-stimulated adipocytes, establishing
that the constitutive internalization of the GIPR is independent of β-arrestin2 (Fig 1D & E).
These data together with the result that β-arrestin1 knockdown did not alter the fraction of
GIPR on the PM (Fig 1B), support a model in which GIPR internalization is independent of
either β-arrestin, an unexpected finding considering the role of β-arrestins in internalization
of other GPCRs.
The amount of GIPR in the PM is dynamically maintained by internalization and recycling.
Since β-arrestin2 depletion abrogates GIPR downregulation without affecting
internalization, we hypothesized a role for β-arrestin2 in regulation of GIPR recycling. The
HA-GIPR-GFP recycling rate constant was measured using an assay adapted from studies of
Glut4 trafficking (Karylowski et al., 2004). In agreement with our previous work
(Mohammad et al., 2014), GIP induced an approximate 2.5 fold inhibition of the GIPR
recycling rate constant, a change that underlies GIPR downregulation (Fig 1F & H). β-
Arrestin2 knockdown abolished GIP-induced inhibition of recycling without affecting GIPR
recycling in unstimulated adipocytes (Fig 1G & H). Thus, GIPR is downregulated by a
mechanism involving β-arrestin2-dependent inhibition of recycling, independent of a role
for β-arrestin2 in regulation of GIPR endocytosis.
GRK 2 and 5 are required for GIP-induced downregulation of GIPR
Activated GPCRs are phosphorylated by GRKs, stimulating the recruitment of β-arrestins to
the receptors and thereby regulating β-arrestin control of GPCR trafficking (Claing et al.,
2002). We next undertook studies to determine whether the role of β-arrestin2 in GIPR
trafficking is dependent on GRKs. We transiently silenced the three GRK isoforms
expressed in adipocytes (Fig S1 and SuppTable 2). Individual knockdowns of GRK 2, 5 or 6
did not alter the PM to intracellular distribution of GIPR in unstimulated adipocytes nor
affect GIP-induced GIPR downregulation (Fig 2A). To test for functional redundancy among
these GRKs, we silenced them in pairs. Double knockdown of GRK 2 and 5 abolished GIPR
downregulation, whereas double knockdowns of GRK 2 and 6, or GRK 5 and 6 did not (Fig
2A). Simultaneous knockdown of all three GRKs did not have an effect beyond the double
GRK 2 & 5 knockdown (Fig 2A). These data support a model in which GRK 2 or 5 are
functionally redundant in GIP-mediated GIPR downregulation.
We hypothesized that if GRKs 2 or 5 are required for β-arrestin2 regulation of GIPR
trafficking, then silencing these proteins should phenocopy β-arrestin2 knockdown,
abrogating GIP inhibition of GIPR recycling without affecting GIPR internalization. In
support of our hypothesis, double knockdown of GRKs 2 and 5 abrogated GIP-induced
slowing of GIPR recycling without affecting recycling in unstimulated adipocytes, whereas
silencing of GRKs 2 and 5 did not affect GIPR internalization in either GIP-stimulated or
unstimulated adipocytes (Fig 2B to G). Thus, GRK 2 or 5 and β-arrestin2 are required for
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GIP-induced GIPR inhibition of recycling and therefore they are molecular components of
the machinery responsible for GIPR downregulation. Despite the roles of GRKs and β-
arrestin2 in promoting internalization of other GPCRs, the internalization of GIPR is
independent of GRKs and β-arrestins.
β-Arrestin2 binding to GIPR requires GIP stimulation and GRK 2 or 5
GRK phosphorylation of GPCRs is involved in the recruitment of β-arrestins to the
receptors. Our functional data support a model in which GRK phosphorylation of GIPR
promotes β-arrestin2 recruitment to GIPR and subsequent slowing of GIPR recycling back
to the PM. To further test this model, we probed for β-arrestin2 binding to GIPR by co-
immunoprecipitation. GIP-stimulation promoted β-arrestin2 binding to GIPR consistent with
β-arrestin2 having a role in GIPR trafficking (Fig 3A). In addition, simultaneous silencing of
GRKs 2 and 5 blocked β-arrestin2 recruitment to GIPR (Fig 3B). These data establish that
despite GRK/β-arrestin2 regulating recycling rather than internalization of GIPR, β-arrestin2
recruitment to activated GIPR is dependent on GRK. β-Arrestin1 did not co-
immunoprecipitate with GIPR in GIP stimulated or unstimulated adipocytes, consistent with
the functional data that β-arrestin1 does not have a role in GIPR trafficking (Fig S2).
GIP-induced slowing of GIPR recycling by β-arrestin2-dependent targeting to the TGN
The GIP-induced slowing of GIPR could be achieved by altering the recycling itinerary of
GIPR or by specifically slowing the movement of GIPR without altering the compartments
transited during recycling, as would be the case if β-arrestin2 binding to GIPR directly
slowed exit from a compartment. The two main recycling pathways are return to the PM
from the endosomal recycling compartment (ERC) and recycling through the TGN (Bard
and Malhotra, 2006; Johannes and Popoff, 2008; Maxfield and McGraw, 2004). These two
pathways have distinct cargos. The transferrin receptor (TR) is an example of a client of the
ERC pathway and TGN46 is an example of a client of the TGN recycling pathway (Banting
and Ponnambalam, 1997; Maxfield and McGraw, 2004). The TGN and ERC are poorly
resolved by conventional light microscopy in adipocytes, making it impossible to distinguish
which of these pathways is utilized in GIPR recycling in adipocytes using conventional
microcopy approaches (Blot and McGraw, 2008; Puertollano et al., 2003). We turned to a
detergent-free, immunoadsorption method to probe the effect of GIP on the localization of
the GIPR. This method has been well established in studies of GLUT4 glucose transporter
trafficking in adipocytes (Bruno et al., 2016; Kandror and Pilch, 2006). Briefly, cells are
broken in the absence of detergent to preserve membranes with their embedded proteins. An
antibody against cytoplasmic domain of a protein is used to immunoadsorb membranes
specifically enriched in that protein. Co-adsorbing proteins, determined by Western blotting,
are in the same membranes as the target protein. We used an anti-TGN-46 antibody to
immunoadsorb the TGN (TGN-46-containing membranes) (Fig 4A and B). Although
TGN-46 is concentrated in the TGN, it continually cycles between the TGN and the PM. We
used an amount of anti-TGN-46 antibody that adsorbed approximately 70% of TGN-46 as a
way to bias the adsorption to those membranes in which TGN-46 is concentrated. Both
Syntaxin 6 (Sx-6) and mannose-6 phosphate receptor (M6PR), two proteins enriched in the
TGN, co-adsorbed to about the same extent as TGN-46, whereas the cis-Golgi matrix
protein, GM-130, did not adsorb with the TGN-46 (Fig 4A & B). These data confirm the
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specificity of the immunoadsorption. Little GIPR was co-adsorbed with TGN-46 in basal
adipocytes, whereas a significant amount of GIPR co-adsorbed in GIP-stimulated cells. The
co-adsorption profiles of Syntaxin 6, M6PR and GM-130 were unaffected by GIP
stimulation. These data demonstrate increased localization of activated GIPR in the TGN.
As a complementary approach to investigate GIPR localization, we immunoadsorbed GIPR
membranes and Western blotted for endosomes with TR and the TGN with TGN-46. In
basal and GIP-stimulated adipocytes, both TR and TGN46 were immunoadsorbed with the
GIPR, demonstrating GIPR is in both TR-containing endosomes and in the TGN (Fig 4C).
There were, however, significant quantitative differences in the amounts of TR and TGN46
co-adsorbed between basal and GIP-stimulated adipocytes. GIP-stimulation caused a
reduction of TR and a concomitant increase in TGN46 immuno-adsorbed with GIPR (Fig
4D). These data demonstrate a redistribution of GIPR from membranes enriched in TR to
those enriched in TGN-46 following GIP-stimulation, establishing an altered intracellular
GIPR trafficking itinerary in stimulated adipocytes correlates with the slowing of recycling.
Targeting to the TGN from endosomes could be through one of the several retrograde
pathways. One of the major pathways depends on sytaxin-6 (Sx-6). Transient knockdown of
Sx-6 blunted the GIP stimulated GIPR downregulation (Fig 4E and Fig S3). These data
support the conclusion that GIP stimulates GIPR targeting to the TGN. These data also
suggest that GIPR targeting to the TGN is via Sx-6 dependent retrograde transport.
To explore the functional consequences of the redistribution, specifically whether the change
in GIPR recycling itinerary accounts for the slow recycling following GIP stimulation, we
measured the recycling rate constants of the ERC and TGN pathways in adipocytes. We used
a Furin-Tac chimera for analyses of the TGN pathway. Furin is a TGN resident
transmembrane protease that cycles between the TGN and the PM, and the Furin-Tac
chimera has been used to quantify trafficking from the TGN (Voorhees et al., 1995). The
recycling rate constant of the ERC pathway (TR recycling) was about 3 times faster than the
TGN pathway (Furin-Tac recycling) (Fig 4F). GIP stimulation did not affect the recycling
rate constants of either the TR or Furin-Tac. The GIP-induced redistribution of GIPR from
the more rapid TR recycling pathway to the slower TGN pathway can account for the GIP-
induced slowing of GIPR recycling.
We developed a simple, 2-pool kinetic model based on GIPR recycling from the ERC or
from the TGN, and modeled changes in the rate of GIPR trafficking from ERC to the TGN
(Fig 4G). The rate constants of this 2-pool model are: ‘Ki’ (internalization from the PM),
kERC (recycling rate from the ERC to PM, TR recycling pathway), kTGN (recycling rate
from the TGN to PM, TGN-36 recycling pathway) and ksort (pathway from the ERC to
TGN) (materials and methods). A fit of the data from Fig 1F yielded a value of ksort (GIPR
ERC to TGN rate) in the basal state of near 0 (with an upper limit value of 0.02 per min, Fig
S4) that increased to a value of approximately 0.22 min per min in GIP-stimulated
adipocytes, revealing a pronounced stimulation of GIPR transit from the ERC to the TGN in
GIPstimulated adipocytes (Fig 4H).
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The model predicts nearly 60% of GIPR on the PM of basal adipocytes, which is reduced to
45% upon GIPR stimulation (Fig 4I), which is in good agreement with the approximate 30%
GIP-induced downregulation of GIPR experimentally determined (e.g., Fig 1B). In fact,
using the values estimated in this study, the maximum drop in surface GIPR is predicted to
be ~30% (equation 11, supplementary materials and methods). In good agreement with the
observed downregulation of PM GIPR measured, indicating that GIP induces the maximum
downregulation achievable by the switch in recycling pathways. The kinetic model also
predicts nearly all of the intracellular GIPR in basal adipocytes is in the ERC, whereas upon
GIP stimulation about 60% of GIPR is in the TGN (Fig 4I). The large redistribution of GIPR
to the TGN predicted by modeling is in agreement with the experimentally measured
increased co-localization of GIPR and TGN46 in stimulated adipocytes (Fig 4A and D).
We hypothesize that the GIP-induced slowing of GIPR recycling requires β-arrestin2
recruitment to GIPR and subsequent rerouting of GIPR from the endosomal to TGN
recycling pathway. This model predicts silencing of β-arrestin2, which abrogates GIP-
induced slowing of recycling, will also inhibit the rerouting of GIPR from endosomes to the
TGN. This is indeed the case, GIPR was not rerouted from TR-containing endosomes to the
TGN in β-arrestin2 knockdown adipocytes, establishing a role for β-arrestin2 in GIP-
induced targeting of GIPR to the TGN (Fig 4J & K).
The carboxyl cytoplasmic domain of GIPR is required for GIP-induced downregulation
The requirement for GRK 2 or 5 in β-arrestin2-dependent downregulation of GIPR suggests
a role for receptor phosphorylation. A homology model based on the glucagon receptor (also
a secretin GPCR) predicts the cytoplasmic domain of GIPR extending from residues 400–
466. Several serines in the predicted C-terminal domain are conserved among different
species, suggesting potential functional relevance of these conserved serines (Fig 5A). To
determine the role of the cytoplasmic domain in GIPR trafficking, we characterized the
behavior of a HA-GIPR-GFP in which the 23 carboxyl-terminal amino acids were deleted
(GIPRΔ444–460). This deletion construct was distributed between peri-nuclear
compartment and the PM, similar to the full length GIPR (Fig 5B). Furthermore, this
deletion did not quantitatively affect the distribution of GIPR between the PM and
intracellular compartments in unstimulated adipocytes, demonstrating that the constitutive
basal internalization and recycling of GIPR is not dependent on the deleted sequences (Fig
5C). GIPRΔ444–460 was not downregulated by GIP stimulation, establishing a role for the
carboxyl-terminal 23 amino acids in downregulation (Fig 5C). GIP stimulation of
GIPRΔ444–460 activated adenylate cyclase to the same degree as WT GIPR, demonstrating
the truncation does not abrogate GIP binding or activation of the receptor (Fig 5D).
Five potential serine phosphorylation sites are disrupted by the deletion, the 4 serines within
the deleted sequences and serine at position 443 at the junction of the deletion (Fig 5A).
Simultaneous mutation of these 5 serines to alanines (GIPR-5A) did not affect GIP-
stimulated receptor downregulation, establishing that the deletion of the last 23 amino acids
of GIPR had an effect beyond the loss of these potential phosphorylation sites (Fig 5E).
Downregulation was impacted when the conserved serine at position 435 was mutated to
alanine in the context of the GIPR-5A mutant (GIPR-6A), although mutation of serine 435
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to alanine by itself did not affect downregulation (Fig 5E). These data are consistent with
GIPR phosphorylation having a role in receptor downregulation while also demonstrating
redundancy (or complexity) in the phosphorylation of GIPR required for downregulation.
GIPR-Gln354 variant is downregulated by redistribution to the TGN recycling pathway
We have previously shown differences in the post-activation trafficking of a naturally
occurring human variant of the GIPR, GIPR-Gln354, in which glutamine is substituted for
glutamate at residue 354 (Mohammad et al., 2014). GIP-stimulated downregulation of the
variant is more pronounced than that of GIPR-Glu354 (50% versus 30%, respectively) and
the time to restore pre-stimulated levels of GIPR following removal of GIPR is 4 times as
long for the GIPR-Gln354 variant. These differences are due to an enhanced GIP-induced
slowing of the recycling of the GIPR-Gln354 variant compared to the slowing of recycling
of GIPR-Glu354 (Mohammad et al., 2014).
GIP-stimulated downregulation of the GIPR-Gln354 variant corresponds to a switch in
recycling pathways, demonstrated by increased co immunoadsorption with TGN46 and a
corresponding decrease in co immunoadsorption with TR upon GIP stimulation (Fig 6A and
B). Thus, despite the enhanced downregulation of the GIPR-Gln354 variant, it is
downregulated by the same mechanism as GIPR-Glu354, which involves GIP-stimulated
targeting to the TGN recycling pathway. Consequently, the enhanced downregulation and
prolonged intracellular sequestration of the variant are not explained by differences in the
intracellular itineraries of GIPR and the GIPR-Gln354 variant.
GIPR-Gln354 variant downregulation is dependent on β-arrestin2 yet independent of GRKs
As is the case for the GIPR-Glu354, β-arrestin2 was required for the GIP-induced
downregulation of the GIPR-Gln354 variant, and knockdown of β-arrestin1 did not affect
GIP-stimulated or unstimulated GIPR-Gln354 variant trafficking (Fig 6C). However, unlike
GIPR-Glu354, downregulation of GIPR-Gln354 variant was not dependent on GRKs, as
simultaneous knockdown of the 3 isoforms expressed in adipocytes did not affect GIP-
stimulated downregulation (Fig 6D). In agreement with GRK-independent downregulation
of the GIPR-Gln354 variant, deletion of the 23 carboxyl-terminal amino acids did not affect
downregulation (Fig 6E). Thus, GIP binding is sufficient to lead to the downregulation of the
GIPR-Gln354 variant independent of GRK phosphorylation of the receptor.
β-Arrestin2 bound to the GIPR-Gln354 variant, as expected based on the effect of β-
arrestin2 depletion on downregulation (Fig 6F). However, significant β-arrestin2 also co
immunoprecipitated with the variant in unstimulated adipocytes, demonstrating β-arrestin2
binding to the variant in the absence of GIP stimulation (Fig 6F and G). To directly establish
that in unstimulated adipocytes there was enhanced β-arrestin2 binding to the GIPR-Gln354
variant, relative to GIPR-Glu354, we directly compared co-immunoprecipitations on the
same gel (Fig 6H and I). There was a ~3 fold increase in β-arrestin2 co-immunoprecipitated
with the variant relative to GIPR. However, basal β-arrestin2 binding did not target the
GIPR-Gln354 variant to the TGN in unstimulated adipocytes (Fig 6B).
Knockdown of GRKs 2 & 5, which did not block GIP-induced downregulation of the GIPR-
Gln354 variant (Fig 6C), did not reduce GIP-stimulated β-arrestin2 binding to the variant
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(Fig 6J and K). These data establish GIP-dependent and phosphorylation-independent
recruitment of β-arrestin2 to the Gln354 variant. Thus, a difference between the predominant
form of GIPR and the GIPR-Gln354 variant is that binding of β-arrestin2 to the variant is
not controlled by phosphorylation of the receptor.
We have previously shown non-typical trafficking behavior of GIPR (Mohammad et al.,
2014). Namely, GIPR is constitutively internalized and recycled back to the PM in both
unstimulated and GIP-stimulated adipocytes, cells that natively express GIPR. GIP promotes
downregulation of GIPR from the PM by slowing GIPR recycling with not effect on
internalization. The trafficking of GIPR has been previously studied ectopically expressed in
a human embryonic kidney cell line (HEK cells), a cell type that does not natively express
GIPR (Ismail et al., 2015; Tseng and Zhang, 2000; Wheeler et al., 1999). In HEK cells, GIP
stimulation resulted in a rapid redistribution of GIPR from the PM to lysosomes (Ismail et
al., 2015). The differences in those results and ours likely reflect the cell types used for
study.
Here we have demonstrated that GIPR downregulation is achieved by a mechanism
involving a pronounced GIP-induced redirection of GIPR from the rapid ERC recycling
pathway to the slower TGN recycling pathway (Fig 7). This change in recycling kinetics
causes a reduction in the steady state PM levels of GIPR. The amount of GIPR on the PM is
controlled by GIP modulation of which of the two constitutive recycling pathways is used,
without GIP affecting the kinetics of those pathways. The perinuclear endosomal and TGN
compartments in the fat cells are poorly resolved by conventional light microscopy. Due to
this limitation we have been unable to use microscopy to co-localize GIPR in these
compartments. Instead we have used established biochemical method of immunoadsorption
of the TGN or GIPR compartments to show that the GIPR indeed traffics to the TGN
compartment upon GIP stimulation. Kinetic modeling revealed that the large GIP-induced
redistribution of GIPR to the TGN pathway is sufficient to account for the measured 30%
reduction on PM GIPR.
Other GPCRs traffic through the TGN and therefore the TGN may be a common site of
GPCR sequestration. For example, activated CC chemokine receptor 5 (CCR5), somatostatin
2A receptor and β1-adrenergic receptor are all targeted, after ligand activation of
internalization, to the TGN (Cheng and Filardo, 2012; Csaba et al., 2007; Escola et al.,
2010). Delivery of GPCRs to the TGN may facilitate the transport of internalized GPCRs to
different final destinations. For example, CCR5 and somatostatin 2A receptors, like the
GIPR, are returned from the TGN to the PM, whereas the β1-adrenergic receptor is targeted
from the TGN for degradation.
Knockdown of β-arrestin2 abrogates targeting of GIPR to the TGN and consequently
inhibits downregulation of the receptor (Fig 1). Binding of β-arrestin2 to the predominant
isoform of the human GIPR, Glu354, is dependent on GRK 2 or 5, consistent with β-
arrestin2 recruitment requiring phosphorylation of GIPR (Fig 3). This redundancy in GRKs
is not unexpected because GRKs are promiscuous with respect to substrate specificity
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(Gurevich et al., 2012; Violin et al., 2006; Zidar et al., 2009). Consistent with a role for
phosphorylation, deletion or mutation of a group of serines in the carboxyl cytoplasmic
domain of the GIPR blocked GIP-stimulated downregulation. Binding of β-arrestins to
activated GPCRs requires more than one phosphorylation site within the receptor (Tobin,
2008). The requirement for multiple phosphorylation sites coupled with the promiscuity of
GRKs in phosphorylation site specificity provides an explanation as to why mutation of
multiple serines in the cytoplasmic domain of GIPR is required to inhibit downregulation
(Fig 5).
β-Arrestins have a role in intracellular trafficking of other GPCRs (Kang et al., 2014;
Magalhaes et al., 2012), including β-arrestin2-dependent sequestration of β1-adrenergic
receptor in the TGN (Cheng and Filardo, 2012). The molecular mechanisms underlying β-
arrestins’ regulation of intracellular trafficking have not been fully described. β-Arrestin2
binds to a number of proteins involved in the control of membrane protein trafficking,
including clathrin, clathrin adaptor complex AP-2, and ARF6 (Kang et al., 2014). Although
future studies are required to define the molecular mechanism of GIPR targeting to the
TGN, the most likely scenario is that β-arrestin2 serves as an adaptor to link GIPR to
machinery that mediate trafficking from ERC to the TGN, with GIPR in unstimulated cells,
when β-arrestin2 is not bound, returned to the PM by the ERC recycling pathway.
The Gln354 variant is associated with altered blood glucose and insulin following glucose
ingestion (Nitz et al., 2007), an increased body mass index, impaired glucose homeostasis in
obese children (Sauber et al., 2010) and reduced bone mineralization density (Torekov et al.,
2014). GIP-stimulated downregulation of the naturally occurring human GIPR-Gln354
variant involves β-arrestin2-dependent targeting to the TGN (Fig 6). Unexpectedly, targeting
of GIPR-Gln354 variant to the TGN and its downregulation are independent of GRKs (Fig
6B). In agreement with downregulation of the GIPR-Gln354 variant being independent of
receptor phosphorylation, deletion of the cluster of serines in the carboxyl cytoplasmic
domain of the variant did not affect downregulation, whereas this deletion in GIPR-Glu354
inhibited downregulation (Figs 5C & 6C). These data suggest a model in which the Gln354
substitution alters the conformation of GIPR in a way that allows for GIP-induced, β-
arrestin2-dependent targeting to the TGN independent of GRK phosphorylation of the
receptor (Fig 7).
In unstimulated adipocytes, β-arrestin2 binding to GIPR-Gln354 is greater than binding to
GIPR-Glu354, (Fig 6I). However, β-arrestin2 binding to GIPR-Gln354 in the absence of GIP
is not sufficient to target the receptor to the TGN (Fig 6A). It has recently been shown that
different GPCRs impose distinct conformations on bound β-arrestin2 (Lee et al., 2016;
Thomsen et al., 2016). These differences in conformations correlate with differences in β-
arrestin2-dependent signal transduction and or the stability of β-arrestin2 binding to the
GPCR. The GPCR-specific conformation of β-arrestin2 may account for how different
GPCRs use β-arrestin2 as a common effector for distinct outputs. Not only can different
GPCRs have different effects on β-arrestin2, but structurally distinct ligands binding to the
same GPCR can also induce different β-arrestin2 conformations (Lee et al., 2016). Our
functional data suggest different conformational states of β-arrestin2 bound to GIPR-Gln354
depending on whether or not GIP is also bound to the receptor. By increasing β-arrestin2
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association with GIPR in the absence of GRK phosphorylation, the Gln354 substitution
might prolong the β-arrestin2 association. This is in principle similar to observations that
switching carboxyl domains of GPCRs can lead to enhanced β-arrestin binding and altered
GPCR trafficking (Thomsen et al., 2016). β-Arrestin2 binding to GIPR-Glu354 (the
predominant isoform) is phosphorylation dependent and it will be reversed by
dephosphorylation, reversing the effects of β-arrestin2 on GIPR behavior. Following GIP
removal, GIPR-Glu354 repopulates the PM within 60 min, whereas it takes 4 hrs for the
Gln354 variant to be restored to pre-stimulation levels (Mohammad et al., 2014). A
consequence of β-arrestin2 binding to the variant independent of phosphorylation is that de-
phosphorylation is not a means to accelerate the reversal of the β-arrestin2 effect on GIPR
trafficking, thereby providing a mechanism for the enhanced downregulation and prolonged
desensitization of the variant GIPR (Mohammad et al., 2014). Consistent with this proposal
that the Gln354 substitution has an effect on GIPR trafficking beyond targeting to the TGN,
kinetic modeling reveals a 30% maximum downregulation by a switch in recycling
mechanism, whereas the variant is downregulated by 50% (Mohammad et al., 2014).
There are at least two retrograde pathways to the TGN, one from the endosomal recycling
compartment and the other from late endosomes (Bard and Malhotra, 2006; Johannes and
Popoff, 2008; Maxfield and McGraw, 2004). The dependence of GIPR downregulation on
Sx-6 (Fig 4E) suggests that GIPR is sorted to the TGN by a sytaxin-6 dependent retrograde
pathway from endosomal recycling compartment.
Glu354 of the GIPR, a class ‘B’ GPCR, is a structural counterpart to a Trp that is an element
of an “activation micro-switch” in class ‘A’ GPCRs (Cordomi et al., 2015). The micro-
switch in class A receptors links transmembrane domains (TM) 6, 7 and 2 (Cordomi et al.,
2015; Xue et al., 2015). A change in the conformation of this micro-switch is important in
activation of class A GPCRs (Cordomi et al., 2015; Franco et al., 2016). In GIPR, Glu354
(TM 6), Ser381 (TM 7) and Arg183 (TM 2) are structural correlates of the micro-switch,
suggesting this region is important in activation of the GIPR (Cordomi et al., 2015). The
naturally occurring Gln354 variant change in the micro-switch biases the GIPR to a
conformation with enhanced β-arrestin2 binding and altered post-activation behavior of the
receptor without activating the receptor. The altered behavior of the variant is conceptually
similar to effects of biased agonists in which structurally distinct ligands binding to the same
GPCR can, through different structural changes, bias signaling output. In the case of the
GIPR-Gln354, a substitution in a region predicted to be important for conformational
changes linked to activation, bias the GIPR structure towards binding of β-arrestin2
independent of GRK-dependent phosphorylation. Different substitutions at position 354
have different effects of the conformation of GIPR, emphasizing the importance of this site
in GIPR behavior. The natural Gln354 substitution affects the conformation of GIPR in a
way that results in recruitment of β-arrestin2 without constitutively activating the receptor
(Mohammad et al., 2014), whereas site-directed mutagenesis to an alanine at 354 results in a
constitutively active receptor (Cordomi et al., 2015).
Neither β-arrestin1 or 2, or any of the three GRKs expressed in cultured adipocytes were
required for internalization of GIPR in basal or GIP-stimulated adipocytes. Although we
only achieved a 65% knockdown of β-arrestin2 and therefore residual β-arrestin2 could
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mediate the internalization, we think this is highly unlikely since knockdown of GRK 2 and
5 results in a near complete reduction of β-arrestin2 binding to GIPR without an effect on
internalization (Figs 2 & 3). The most parsimonious interpretation of the data is that GIPR is
internalized by a constitutive, non-specialized endocytic mechanism in both unstimulated
and GIP-stimulated adipocytes. The internalization rate constant of GIPR was unchanged by
GIP stimulation; consistent with the same internalization mechanism being used in both
unstimulated and stimulated adipocytes. The biological significance of GRK and β-arrestin
independent internalization is not clear at this time.
Cell culture, transfection, and electroporation
3T3-L1 fibroblasts were used after differentiation into adipocytes, and electroporated as
described previously (Mohammad et al., 2014).
Quantitation of GIPR downregulation
HA-GIPR-GFP was transiently expressed in 3T3-L1 adipocytes by electroporation. The
following day, the cells were serum starved for two hours prior to the GIP treatment. Cells
were then either treated with 100 nM GIP for one hour (stimulated) or incubated without
GIP (basal) for one hour. The cells were immediately cooled on ice and fixed with 3.7%
formaldehyde. The surface GIPR was stained with anti-HA antibody followed by a Cy3
labeled secondary antibody. The cells were imaged for GFP and Cy3 staining. GFP was used
to identify cells expressing the GIPR. The GFP and Cy3 fluorescence was measured for cells
expressing GFP (HA-GIPR-GFP) being unbiased to Cy3. Surface associated GIPR was
measured as an average of the Cy3:GFP ratio (surface staining normalized to total GIPR
expression) for each cell. A minimum of 30 cells was counted. GIPR downregulation was
quantified as a decrease in Cy3:GFP upon GIP treatment. Statistical significance was
measured for GFP expression and Cy3 staining in cells within each dish (making sure of
insignificant variation in expression and staining and identifying outliers). Statistical
significance was also measured for all cells among dishes to ensure GIPR downregulation,
and between
experiments.
GIPR exocytosis
Cells transiently transfected with HA-GIPR-GFP used. Incubated with or without 100 nM
GIP for 60 min. Cells were then incubated with anti HA antibody for times ranging from 5
min to 60 min. 100 nM GIP was included for GIP treatment group. Cells were cooled
immediately, fixed and stained with Cy3 secondary after permeabilization. Cell associated
anti-HA antibody was quantified as Cy3 fluorescence. Cy3 normalized to GFP (Cy3:GFP)
was plotted against time. The data were fit to a single exponential rise to derive an apparent
exocytic rate constant.
GIPR internalization
Cells electroporated with HA-GIPR-GFP were used. Incubated with or without 100 nM GIP
for 60 min. Cells were then incubated in anti-HA antibody with or without GIP for different
times from 2 min to 15 min., at the end of each time point, the cells were immediately
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cooled and fixed. The surface bound antibody was stained with Cy5 secondary in non-
permeabilized cells. The internalized antibody was stained with Cy3, after permeabilization.
The internalized anti-HA antibody was quantified at each time point as Cy3 fluorescence.
Cy3:Cy5 was plotted against time, fitted on the equation for straight line. The slope of the
line was calculated as the apparent rate constant of internalization.
Stable expression of HA-GIPR-GFP in adipocytes
HA-GIPR-GFP stable cells were generated by using the ViraPower lentiviral expression
system (Life Technologies).
HA-GIPR-GFP co-immunoprecipitation
The method was adapted from (Bruno et al., 2016) for the immunoadsorption of GLUT4
membranes.
HA-GIPR-GFP Immunoadsorption
Cells stably expressing HA-GIPR-GFP were differentiated into adipocytes, treated with or
without GIP and lysed in buffer containing 20 mM HEPES, 1 mM EDTA, 250 mM sucrose,
and protease inhibitors and no detergent to prevent membranes from solubilizing. The lysate
was cleared by centrifugation at 700xg and incubated with anti-GFP microbeads. The lysate
containing the beads was loaded on the μMACS magnetic column. HA-GIPR-GFP
membranes were eluted after washing the column and elution in 1X lammeli buffer. The
lysate (input) flow through (unbound) and elution (bound) were run in SDS-PAGE and
blotted for HA-GIPR-GFP, TGN46 or TR.
Data acquisition and processing
Fluorescent images were collected on a DMIRB inverted microscope (Leica Microsystems,
Deerfield, IL), using a 20X objective. Fluorescence quantifications were done using
MetaMorph image processing software (Molecular Devices, Sunnyvale, CA), as described
previously (Blot and McGraw, 2008; Bruno et al., 2016; Mohammad et al., 2014; Sadacca et
al., 2013).
Kinetic model derivation and description
The kinetic model has been described in supplementary methods.
Statistical analysis
Statistical significance was calculated by Student’s t-test.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
This work was supported by DK096925 (TEM). We thank members of the McGraw lab for helpful discussions.
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. βA2 is required for GIP-stimulated GIPR sequestration and slowed recycling
(A) Fixed non-permeabilized adipocytes expressing HA-GIPR-GFP, with or without β-
arrestin1 (βA1) or β-arrestin2 (βA2) knockdown (KD). Surface GIPR was stained with Cy3
using anti HA-epitope antibody. Epi fluorescence images. Scale bar: 50μm
(B) Quantitation of GIPR plasma membrane to total cell expression (PM-to-Total)
distribution in basal and GIP-stimulated (100 nM, 60 min) cells. Adipocytes were
electroporated with HA-GIPR-GFP and with no siRNA (WT), βA1, βA2 or βA1+2 siRNA.
PM-to-total (Cy3/GFP) were determined as described in materials and methods. Data from
individual experiments are normalized to the control cells in basal conditions. Data are
averages of 9 independent experiments ± SD., P ≤ 0.05.
(C) GIPR internalization in WT adipocytes was measured as described in materials and
methods. The slopes, which are the rate constant of internalization, are plotted in panel E.
Data are averages ± SD of 9
independent experiments.
(D) GIPR internalization experiment in βA2 KD adipocytes. Data are averages ± SD of 9
independent experiments. Each βA2 KD experiment was accompanied by an experiment in
WT adipocytes (shown in (C)).
(E) Internalization rate constants (Ki) for GIPR internalization in WT or βA2 KD
adipocytes. The Ki were calculated as slopes of straight lines from (C) and (D). Data are
averages of 9 independent experiments ± SD.
(F) GIPR Exocytosis was measured as described in materials and methods. Cell associated
Cy3 normalized to GFP was plotted against time. Data are average ± SD of 9 independent
experiments.
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(G) GIPR exocytosis experiment in βA2 KD adipocytes. Data are averages ± SD of 9
independent experiments. Each βA2 KD experiment was accompanied by an experiment in
WT adipocytes (shown in (F).
(H) Exocytic rate constants (Ke) for GIPR in WT or βA2 KD cells calculated from (F) and
(G). The data were fit to a single-phase exponential rise equation. Data are averages of 9
independent experiments ± SD., p<0.05.
See also Fig S1.
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. GRKs 2 or 5 are required for GIP-stimulated GIPR sequestration and slowed recycling
(A) Quantitation of GIPR PM-to-Total distribution in basal and GIP-stimulated WT (no
siRNA), and GRK KD adipocytes. Data are averages of 5 independent experiments ± SD.,
p≤0.05.
(B) GIPR Exocytosis experiment in WT adipocytes. Data are averages ± SD of 5
independent experiments.
(C) GIPR exocytosis in GRK2+5 double KD adipocytes. Data are average ± SD of 5
independent experiments. Each GRK 2+5KD experiment was accompanied by an
experiment in WT adipocytes (shown in panel B).
(D) Exocytic rate constants (Ke) for GIPR in WT or GRK2+5 KD cells calculated from (B)
and (C). The data were fit to a single phase exponential rise equation. Data are averages of 5
independent experiments ± SD., p<0.05.
(E) GIPR internalization in WT adipocytes. The internalization rate constants are plotted in
panel G. Data are averages ± SD of 7 independent experiments.
(F) GIPR internalization in GRK2+5 double KD adipocytes. Data are averages ± SD of 7
independent experiments. Each GRK2+5 double KD was accompanied by an experiment in
WT adipocytes (shown in panel E).
(G) Internalization rate constants (Ki) for GIPR internalization in WT or βA2 GRK2+5
double KD. The Ki were calculated as slopes of straight lines from (E) and (F).
See also Fig S1 and S2.
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. βA2 binding to GIPR is GIP- and GRK2/5-dependent
(A) Immunoblot for co-immunoprecipitated HA-GIPR-GFP blotted for HA-GIPR-GFP and
βA2. IP was done with or without GIP stimulation. (Right) Quantification of co
immunoprecipitation experiments like that shown in the left panel. βA2 in each lane was
normalized to its input and to the GIPR in the IP. Data are averages ± SD of 3 independent
experiments.,
P<0.05.
(B) Immunoblot for co-IP of HA-GIPR-GFP in GRK 2+5 KD cells. Blot was done for HA-
GIPR-GFP and βA2. (Right) Quantification of co immunoprecipitation experiments like that
shown in the left panel. βA2 in each lane was normalized to its input and to the GIPR in the
IP.
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. GIPR is localized to TR-containing endosomes in basal conditions and to TGN46-
containing membranes upon GIP stimulation
(A) Immunoblot of TGN-46 immunoadsorption from basal and GIP stimulated adipocytes.
TGN-46 membranes were immunoisolated using anti-TGN-46 antibody in the absence of
detergent. The Input (In), unbound flow-through (FL) and elution (Bd) were run on SDS-
PAGE and blotted for TGN markers Syntaxin 6 and mannose-6-phosphate receptor (M-6-P
cation independent) and cis-golgi marker GM-130 and for GIPR.
(B) Quantification of (A). Relative enrichment of proteins in elution were calculated by
normalizing to the flowthrough.
(C) Immunoblot of GIPR immunoadsorption from basal and GIP stimulated WT adipocytes.
GIPR-GFP membranes were pulled down in absence of detergent. The elution (bound) was
run on SDS-PAGE together with input and unbound (FL) and blotted for GIPR, TR and
TGN-46.
(D) Quantification of (C). Relative amounts of TR or TGN in the elution were calculated by
normalizing to input and GIPR in the IP.
(E) Quantification of GIP induced GIPR downregulation in syntaxin-6 (Sx-6) knockdown
adipocytes. Two different siRNAs were used. Both knockdowns show a decrease in GIPR
downregulation showing that it depends on Sx-6. Data are averages ± SD of 3 independent
experiments., P<0.05.
(F) Exocytosis rate constants for TR and TGN vesicles compared with GIPR in basal and
GIP stimulated condition. GIPR ke values are plotted from data in Fig. 1.
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(G) Schematic representation of GIPR recycling from the ERC (TR-containing endosomes)
or TGN compartments with associated rate constants. ki, internalization rate constant,
‘kERC’, rate from the ERC; ‘kTGN’, rate from TGN; and ‘ksort’, GIPR sorting rate from ERC
to the TGN. Additional connectivities between these compartments did not significantly
improve the performance of this model.
(H) Calculation of GIPR sorting rate constant (Ksort) by using equation 1 (supplementary
methods) and the model in Fig. 4G. Data from Fig. 1F was used. Inset shows the value of ‘r’
calculated under basal or upon GIP stimulation.
(I) GIPR as a fraction of total in different compartments under basal or GIP stimulated
conditions. The values were calculated from (F).
(J) And (K) Immunoblot of GIPR immunoadsorption from basal and GIP stimulated βA2
KD adipocytes.
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. GIPR intracellular domain is required for GIP induced GIPR sequestration
(A) Amino acid sequence of GIPR predicted cytoplasmic domain (residues 400 to 466). The
C-terminal deletion is marked by the red bar. Serines mutated to alanines are noted by
arrows. Serines conserved in human, mouse, rat and bovine GIPR are shown in green. The
sequence also contains two threonines as potential phosphorylation sites. However, these
threonines are not conserved in mammals.
(B) Images of GIPRΔ444-466-GFP construct in basal adipocytes. Plasma membrane GIPR
was labeled by indirect immunofluorescence with anti-HA/Cy3 secondary antibody of fixed
cells. Scale bar: 50μm.
(C) Quantification of downregulation of GIPRΔ444-466-GFP constructs upon GIP
stimulation. The data are the PM-to-Total of GIP-stimulated normalized to the basal PM-to-
Total., P<0.05.
(D) GIP-stimulated cAMP production in adipocytes expressing GIPR or GIPRΔ444-466.
The endogenous GIPR was knocked down by siRNA such that cAMP production was
downstream of the ectopically expressed GIPRs.
(E) GIP induced downregulation of the GIPR serine mutants. For each construct, the
downregulation has been plotted as PM-to-Total normalized to their basal. The unstimulated
level (basal) is shown with a dashed line.
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. Redistribution of the GIPR-Gln354 variant to the TGN is dependent on GIP and βA2 and
independent of GRKs
(A) HA-GIPR-Gln354-GFP Immunoadsorption from WT adipocytes and blotted for GIPR,
TR and TGN46.
(B) Quantification of data from experiments like that shown in panel A. TR and TGN46 in
elution were calculated as normalized in total (input) and GIPR in the elution. Data are
average ±
SD from 3 independent experiments.
(C) Effect βA1 or βA2 KD on PM-to-Total distribution of GIPR-Gln354 variant. Data are
normalized to their own basal values. Data are averages ± SD of 3 independent experiments.,
P<0.05.
(D) Effect of GRK2+5+6 triple KD on PM-to-Total distribution of GIPR-Gln354 variant.
Data are averages ± SD of 3 independent experiments., P<0.05.
(E) PM-to-Total distribution of GIPR-Gln354 variant and GIPR-354Gln-Δ444-466 deletion
mutant. Data are averages ± SD of 3 independent experiments., P<0.05.
(F) Co-immunoprecipitation of βA2 with GIPR-Gln354.
(G) Quantification of data from experiments like that shown in panel F. Data are average ±
SD from 3 independent experiments.
(H) Relative βA2 binding to GIPR and GIPR-354Gln. GIPR and GIPR-354Gln Co-IP were
blotted for GIPR and βA2 on the same blot.
(I) Quantification of data from experiments like that shown in panel H. Data are average ±
SD from 3 independent experiments.
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(J) GIPR-354Gln-GFP:βA2 binding in GRK2+5 double KD cells.
(K) Quantification of data from experiments like that shown in panel H. Data are average ±
SD from 3 independent experiments.
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. Schematic of the GIPR trafficking pathway
(A) GIPR is recycled constitutively by the fast TR recycling pathway (black arrows). Upon
GIP stimulation, the GIPR is sorted to the slower TGN pathway (blue arrows). This
redistribution of recycling pathways results in a dynamic reduction of GIPR from the plasma
membrane. The redistribution of GIPR from TR to TGN pathway is regulated by β-arrestin2
(βA2) binding, which in turn is recruited in response to ligand binding and GRK mediated
phosphorylation.
(B) Downregulation of the GIPR-354Gln variant is by a similar dynamic sequestration
mechanism, requiring GIP stimulation and βA2 targeting of the variant from the TR to the
TGN recycling pathway. However, βA2 binding to the variant is independent of GRK
phosphorylation.
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