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It has been widely assumed that the production of the ubiquitous second messenger cyclic AMP, which is mediated by cell
surface G proteincoupled receptors (GPCRs), and its termination take place exclusively at the plasma membrane. Recent studies reveal that diverse GPCRs do not always follow this conventional paradigm. In the new model, GPCRs mediate G-protein
signaling not only from the plasma membrane but also from endosomal membranes. This model proposes that following ligand
binding and activation, cell surface GPCRs internalize and redistribute into early endosomes, where trimeric G protein signaling
can be maintained for an extended period of time. This Perspective discusses the molecular and cellular mechanistic subtleties
as well as the physiological consequences of this unexpected process, which is considerably changing how we think about GPCR
signaling and regulation and how we study drugs that target this receptor family.
1
Laboratory for GPCR Biology, Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania,
USA. 2Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA. *e-mail: jpv@pitt.edu
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PERSPECTIVE
Arr
supports the view that the b2AR induces
cAMP through two episodic phases: the
Endosome
first takes place at the plasma membrane
Endosome
Endosome
and is responsible for an acute but short
cAMP response, and the second happens
in early endosomes a few minutes after
Ppase
receptor internalization. This process is
Lysosome
GGTP
compatible with the actions of b-arrestins
that not only uncouple the receptor from
P P
GS but also recruit the cAMP-specific
cAMP
phosphodiesterase (PDE4) to the plasma
membrane29 and engage in the formation
of clathrin-coated pits. The PTHR or the
Figure 1 | Classical versus endosomal signaling models of GPCR. Activation and desensitization of
V2R differ from b2AR, however, because
a cAMP response mediated by GPCRGS systems proceed through a succession of biochemical and
b-arrestins promote rather than attenuate
cellular events that initially take place at the cell membrane and result in the induction, propagation
cAMP production in response to PTH or
and termination of the second messenger molecule (steps 16). In the classical model, GPCRG
vasopressin. The mechanisms by which
protein systems are only active on the cell surface and internalize to be degraded and/or replaced by
this occurs are starting to be undernewly synthesized GPCRs. In the new model, GS and cAMP signaling can continue after internalization
stood and are discussed in the following
of ligandGPCR complexes in endosomes (step 3). The figure is based on ref. 64. Arr, arrestin;
paragraph.
Ppase, protein phosphatase.
Clear evidence that a particular receptor conformation is needed to maintain
laid the foundation for the new model that GS and cAMP signaling GS signaling from subcellular compartment has come from studcan continue after internalization of ligandGPCR complexes in ies on the PTHR, a prototypical GPCR family 2 member that
endosomes (Fig. 1). Parallel studies on the sphingolipid S1P recep- regulates Ca2+ homeostasis by its actions on bone and kidney.
tor (S1P1R) extended this model to the inhibitory G protein (Gi) for As for all GPCRs, the PTHR is likely to exist in a variety of difadenylate cyclases by reporting that internalization of the S1P1R and ferent conformations that are stabilized not only by the type of
its trafficking in the trans-Golgi network contributed to the sustained interacting agonist (full, partial or inverse) but also by interactGi-dependent signaling mediated by FTY720, a S1P1R agonist19. ing signaling proteins3,3034. Recent studies using pharmacological
Initially recognized in 2009 for the PTHR and the TSHR, sustained and biophysical approaches provide new clues as to the nature
GS and cAMP signaling mediated by internalized GPCRs has been of such altered conformational states possible for the PTHR and
further reported for other peptide hormone receptors such as the their relevance to endosomal receptor or Gs signaling and related
glucagon-like peptide 1 receptor (GLP-1R)20, the pituitary adenylate biological actions8,35,36. In the classical GPCR signaling paradigm,
cyclase activating polypeptide (PACAP) type 1 receptor21 and the receptors were thought to exist in a low-affinity ligand-binding
vasopressin type 2 receptor22 (V2R), and it has also been extended to state when uncoupled from G proteins and to shift to a high-affinmonoamine neurotransmitter receptors including the b2-adrenergic ity state only upon G-protein coupling37. Studies on the PTHR
and dopamine D1 receptors23,24, many of which have been recently showed that it deviates from the classical model. It can form
high-affinity complexes with PTH or its N-terminally synthetic
reviewed2527.
analog PTH(134)38,39, even in the absence of G-protein coupling,
as the complexes remain stable in the presence of GTPgS, a guaMechanisms of prolonged signaling at GPCRs
Endosomal GPCR signaling via G proteins incites new ques- nine nucleotide analog that induces receptorG protein dissotions about what mechanisms maintain and regulate G-protein ciation. In contrast, PTHrP, the other native agonist ligand for
signaling from endosomal membranes. A new approach using PTHR40,41, forms complexes that are more like those formed with,
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cAMP response
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RG conformation
a
Binding (%)
[125I]M-PTH(134) + GTPS
PTH(134)
PTHrP(136)
80
60
PTHrP(136)
40
20
0
13 12 11 10 9 8 7 6 5
log[ligand](M)
13 12 11 10 9 8 7 6 5
log[ligand](M)
Bound (%)
100
80
60
Control
Control
40
+ GTPS
20
[125I]PTHrP
0
0
+ GTPS
50
100
Time (min)
c
0
150
[125I]PTH
0
50
100
150
Time (min)
GSND
GSND
0.25
Control
0.5
0.75
Control
1.00
PTHrP dissociation
0
100
Time (s)
200
PTHrP dissociation
300
PTHrP
100
100
200
300
600
900
Time (s)
PTH
80
60
40
20
0
R0 conformation
[125I]M-PTH(115) + GSND
100
Dissociation (F/F0)
Bound (%)
300
600
Time (s)
900
300
Time (s)
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Signal OFF
Tra
n
Tra
n
si e n t
Signal ON
S us
t ai
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Number of steps
ne
703
Sustained cAMP signaling produced by internalized receptors provides new insights into physiological processes as it
may be involved in cardiac neuron excitability regulated by the
PACAP type 1 receptor21 and in insulin secretion by internalized
glucagon-like peptide 1 receptor in pancreatic beta cells. This new
model also explains the physiological bias between two medically
important ligands acting at the V2R, vasopressin and oxytocin,
when used as a therapy for disorders of water and electrolyte
transport22. The divergent antinatriuretic and antidiuretic effects
produced by these ligands, which are either strong (vasopressin)
or weak (oxytocin), are likely to account for the different cAMP
dynamics between vasopressin (sustained endosomal cAMP production) and oxytocin (short cAMP production limited to the
plasma membrane).
The emerging endosomal GPCRGS signaling model invites
us to change our thinking about GPCR signaling and its regulation and move toward new directions to develop ligands that
have improved efficacies for treating diseases by targeting GPCR
in specific cellular locations such as endosomes. In the case of
the PTHR, certain synthetic PTH analogs have been identified
that bind with even higher affinity to R0 than PTH(134)36,42.
This enhanced selectivity for the R0 state of PTHR conformation is accompanied by markedly prolonged cAMP responses from endosomes
a
in cells and, notably, prolonged hypercalb
PTH
LA-PTH
LA-PTH
cemic and hypophosphatemic responses
PTHrP
when injected into mice. One particuPTHR
Oste
larly long-acting PTH analog, which is
obla
AC
Plasma
st
called LA-PTH and consists of a unique
mem
bra
M-PTH(114)/PTHrP(1536)
hybrid
ne
structure (where M = Ala1,12, Aib3, Gln10,
Har11, Trp14 or Arg19), can induce elevacAMP
cAMP
cAMP
tions of serum calcium in mice that persist
Short Intermediate Long
for nearly 24 h following a single subcutasignal
signal
signal
neous injection, which contrasts markedly
Endosome
with injections of PTH(134), which raise
serum calcium for only 24 h42 (Fig. 4).
The prolonged responses of these analogs
in vivo can be explained by their stable
Time
Hypothyroidism
binding to the PTHR in bone and kidney
hypocalcemia
target cells, although a minor contribution
of a low-level of systemic exposure cannot be ruled out. This class of R0-selective
c
PTH analogs is thus of interest as a potential new mode of therapy for patients with
1.6
Control
hypoparathyroidism (a condition that
1.5
PTH
leads to abnormal low levels of ionized calLA-PTH
1.4
cium in blood and thus affects all aspects
1.3
of calcium metabolism), which is now
conventionally treated with vitamin D and
1.2
calcium supplements and for which in vivo
1.1
action of injected PTH(134) is too short
0
2
4
6
8
10
lived, due in part to rapid clearance and
Time after injection (h)
short action on the receptor62. The disease
Figure 4 | Endosomal PTHR signaling: from bench to bedside. (a) Studies in cells led to the
might thus be more approachable with a
discovery that PTH, as opposed to PTHrP, sustains G-protein activity and cAMP production after
long-acting PTH ligand that favors endoPTHR internalization into early endosomes. (b) This observation is changing how we think about
somal PTHR signaling. Understanding
cellular signaling of the PTHR and is motivating the development of PTH analogs able to promote the
its molecular and cellular basis is likely
endosomal cAMP signaling. One of them, LA-PTH, mediates a markedly prolonged cAMP signaling
to lead to important insights into fundaresponse in cells and prolonged hypercalcemic responses when injected into mice. (c) LA-PTH is now
mental mechanisms by which the PTHR
in preclinical development via the US National Institutes of Health BrIDGs program63 for eventual
functions and may potentially lead to new
testing as a future treatment for hypoparathyroidism. Figure adapted from refs. 36, 42.
strategies for PTH drug design. Indeed,
Blood Ca2+ (mM)
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Acknowledgments
This work was supported by the National Institute of Diabetes and Digestive and Kidney
Diseases of the National Institutes of Health under award numbers R01 DK087688 and
DK102495 (to J.-P.V.) and P01 DK11794 (project I to T.J.G.).
Author contributions
J.-P.V., F.G.J.-A. and T.J.G. each contributed to the writing of this manuscript.
Additional information
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