Download G protein-coupled receptors: mutations and endocrine diseases

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G protein-coupled receptors: mutations
and endocrine diseases
Gilbert Vassart and Sabine Costagliola
Abstract | Over the past 20 years, naturally occurring mutations that affect G protein-coupled receptors (GPCRs)
have been identified, mainly in patients with endocrine diseases. The study of loss-of-function or gain-of-function
mutations has contributed to our understanding of the pathophysiology of several diseases with classic
hypophenotypes or hyperphenotypes of the target endocrine organs, respectively. Simultaneously, study of the
mutant receptors ex vivo was instrumental in delineating the relationships between the structure and function of
these important physiological and pharmacological molecules. Now that access to the crystallographic structure
of a few GPCRs is available, the mechanics of these receptors can be studied at the atomic level. Progress in
the fields of cell biology, molecular pharmacology and proteomics has also widened our view of GPCR functions.
Initially considered simply as guanine nucleotide exchange factors capable of activating G protein-dependent
regulatory cascades, GPCRs are now known to display several additional characteristics, each susceptible to
alterations by disease-causing mutations. These characteristics include functionally important basal activity
of the receptor; differential activation of various G proteins; differential activation of G protein-dependent and
independent effects (biased agonism); interaction with proteins that modify receptor function; dimerizationdependent effects; and interaction with allosteric modulators. This Review attempts to illustrate how natural
mutations of GPCR could contribute to our understanding of these novel facets of GPCR biology.
Vassart, G. & Costagliola, S. Nat. Rev. Endocrinol. 7, 362–372 (2011); published online 8 February 2011; doi:10.1038/nrendo.2011.20
Introduction
Institut de Recherche
Interdisciplinaire en
Biologie Humaine et
Moléculaire (IRIBHM),
Faculty of Medicine,
Université Libre de
Bruxelles (ULB),
808 Route de Lennik,
1070 Brussels,
Belgium (G. Vassart,
S. Costagliola).
The G protein-coupled receptors (GPCRs) comprise
one of the largest protein families in both invertebrates
and vertebrates. All GPCRs have a shared structure of
seven transmembrane α helical domains, hence their
other popular name—7TM receptors. For reasons that
are unclear, the three-dimensional structure achieved by
insertion of seven α helices into a cell membrane seems
to be particularly suited to the formation of biological
sensors. 7TM proteins similar to GPCRs first evolved in
prokaryotes as bacteriorhodopsin and sensory rho­dopsin
(light sensors).1 The family of GPCR molecules then
diversified, probably by convergent evolution, into sensors
of both external stimuli (such as light, odors, pheromones
and flavors) and internal stimuli (for example, hormones,
neuropeptides, bioactive amines and lipids, nucleotides
and ions). Humans have ~750 GPCRs, of which ~300 are
nonolfactory in function. The GPCR family is divided
into five subfamilies with little, if any, shared homology in
their primary structure.2 The GPCRs implicated in endocrine functions belong to subfamilies A (prototypes: rhodopsin and β2 adrenergic receptor), B (prototype: secretin
receptor) and C (prototype: metabotropic glutamate
receptors), with the vast majority in family A.
The aim of this Review is to provide an update on the
field of GPCR mutations and endocrine diseases, which
has previously been reviewed elsewhere.3–6 Rather than
Correspondence to:
G. Vassart
[email protected]
Competing interests
The authors declare no competing interests.
362 | JUNE 2011 | VOLUME 7
describing individual diseases in detail, this Review will
illustrate how GPCR mutations, whether known or still
to be discovered, might contribute to our understanding of the diverse facets of GPCRs involved in the field
of endocrinology.
The G protein-coupled receptors
The classic mode of action of GPCRs has been defined
from dissection of the mechanism coupling activation
of the β2 adrenergic receptor to generation of the second
messenger cyclic AMP (cAMP) (Figure 1).7,8 The activated receptors function as guanyl nucleotide exchange
factors (GEFs), with subsequent stimulation of effectors
by the α or βγ subunits of the G proteins. According to
their canonical mode of action, GPCRs could be viewed
as devices that transmit an extremely wide range of signals
through the cell membrane, which results in activation of
a limited number of cytoplasmic regulatory cascades that
are controlled by G proteins.
The crystallographic structure of a handful of GPCRs
belonging to subfamily A has been determined,9–12 providing templates for realistic modeling of members of this
subfamily in their inactive state. A wide range of artificial
and natural mutations in the genes that encode GPCRs
have been studied over the past 20 years.13–15 These studies
led to theories on how GPCRs are activated; however, the
direct structural data to confirm or refute these theori­es
are only just becoming available. 10 The first activating mutation identified in the β2 adrenergic receptor
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suggested that activation resulted from the release of a
structural constraint (or lock) that kept the wild-type
receptor inactive.16,17 Structural data obtained from
studying opsin at low pH (expected to mimic the active
state) point to a mechanism of activation that involves
subtle changes in the conformations of the ligand-binding
pocket.10 These changes are associated with a dramatic
movement of transmembrane helix (TM) 6 secondary
to rupture of a TM3–TM6 ionic lock. The result of this
movement is the opening of a cavity between the cytoplasmic ends of TM3, TM5 and TM6 enabling inter­
action with, and activation of, the G protein.18,19 Crystals
obtained from an agonist-bound β2 adrenergic receptor
stabilized by a camelid antibody (nanobody) that behaves
in a similar way to G protein confirmed and extended
these results. Binding of the agonist or activating mutations are expected to disrupt stabilizing interactions,
thereby lowering the energy barrier and enabling transition of the receptor from the inactive to the active state,
which is capable of interacting with the G protein.20
The initial model of receptor activation involved
the agonist-dependent shift of an equilibrium from an
in­active conformation to an active conformation.21 How­
ever, in addition to this classic view, a series of concepts
that comp­licate the picture must be considered to fully
appreciate the effects of mutations in the genes that
encode GPCRs. We now know that multiple inactive
and active conformations of GPCRs do exist and that
different agonists might stabilize active conformations
of a given receptor with different preferences for G proteins (hence activating different regulatory cascades).22
Initially believed to function only in desensitization
of GPCRs following their activation, β arrestins are
now considered to be molecular scaffolds that control
Gp
­ rotein-independent regulatory cascades.22 Biased
agonism has been defined as the ability of some agonists
to make a given GPCR ‘choose’ between coupling to different G proteins, or between activation of G proteindependent and β arrestin-dependent cascades.22 The
capacity of GPCRs themselves to function as scaffolds,
binding proteins with regulatory or targeting effects, has
also been demonstrated.23,24 These effects mainly involve
the C‑terminal intracellular tail of GPCRs. Many nonmutated GPCRs display basal activity, which makes
them constitutive activators of regulatory cascades, in
addition to sensors of their cognate agonist. GPCRs have
the capacity to homodimerize or heterodimerize25,26 and
their functional characteristics have sometimes been
shown to depend on their quaternary structure.
Endocrinology is the field par excellence in which
the distinction has been made between loss-of-function
(hypo) and gain-of-function (hyper) phenotypes of the
target endocrine organs. Mutations of the GPCRs that are
active in endocrine organs or their target tissues are rare
causes of such phenotypes. The implicit ‘brake or accelerator’ analogy for the pathophysiology of loss-of-function
or gain-of-function mutations, respectively, must now
be altered by consideration of the variety of functional
characteristics displayed by GPCRs in addition to their
coupling to G proteins.
Key points
■■ G protein-coupled receptors (GPCRs) are the largest family of transmembrane
receptors
■■ GPCRs are key factors in endocrinology, as they are the main sensors of the
internal environment
■■ Hereditary and congenital forms of classic endocrine diseases that display
hypophenotyes or hyperphenotypes of the target endocrine organs are
attributable to loss-of-function or gain-of-function mutations of GPCRs,
respectively
■■ In addition to their canonical role as guanine nucleotide exchange factors,
GPCRs have a series of G protein-independent effects that might be the cause
of many endocrine diseases
■■ Endocrine phenotypes resulting from mutations that affect noncanonical
functions of GPCRs remain to be identified
Ligands
Sensory stimuli (light, odor, taste)
ions, neurotransmitters, chemokines,
hormones, prescribed drugs
N
Effectors
Adenylyl cyclases ( )
Phospholipase Cβ ( )
K+, CI–, Na+ channels ( )
VSCC ( )
GIRK ( )
PI3 kinase ( )
GPCR
Extracellular
Membrane
Intracellular
α
C
γ
β
Off
On
α
γ
GTP
β
GDP
G proteins
Gs, Gq, Gi/o, G12/13
cAMP
IP3, DAG
K+ current
PI3 kinase
Figure 1 | Binding of its agonist to a GPCR causes a conformational change that
activates the guanine nucleotide exchange function of the receptor towards one of
the possible interacting heterotrimeric Gαβγ proteins. The result is the
replacement of GDP by GTP on the α subunit, which causes its activation and the
dissociation of the βγ subunits. The activated α subunit, classically considered to
dissociate from the receptor, and the βγ subunit activate downstream effectors.
These effectors are numerous and depend on the nature of the subunits, for
example αs and αi will stimulate and inhibit adenylyl cyclase, respectively; αq will
activate phospholipase C, whereas βγ can for instance activate GIRK channels and
PI3 kinase. The intrinsic GTPase activity of the α subunit causes the system to
return to the inactive state, with GDP bound to a subunit of the reconstituted
Gαβγ heterotrimer. Not illustrated on this scheme is the basal activity displayed by
some receptors and the G protein-independent effects of GPCRs. Abbreviations:
GPCR, G protein-coupled receptor; GIRK, G protein-regulated inwardly rectifying
potassium; PI3, phosphatidyl inositol 3; VSCC, voltage sensitive calcium
channels. Adapted with permission from © Vilardaga, J.-P. et al. J. Cell Sci. 123,
4215–4220 (2010).106
Loss-of-function mutations
Simple loss of function
In endocrinology, loss of GPCR function is associated
with global hypophenotypes of the target tissues; for
example, hypothyroidism, hypogonadism, short stature,
dia­betes insipidus and hypocorticism (Table 1). The clinical aspects of these conditions are convincingly explained
by a decrease in the activity of the GPCR-positive cells,
which is directly related to the severity of the mutation. As
expected for loss-of-function mutations, the corresponding phenotypes are mainly transmitted as autosomal or
X‑linked recessive traits (Table 1). The severity of the
disease might extend over a wide range, depending on
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Table 1 | Loss-of-function mutations of G protein-coupled receptors causing endocrine diseases
Receptor
Disease
Mechanism
Mode of inheritance
Reference
Argine vasopressin receptor 2
Nephrogenic diabetes insipidus
Simple loss of function or
constitutive desensitization
X-linked recessive
Fujiwara & Bichet79
Melanocortin 2 receptor
Familial glucocorticoid deficiency type 1
Simple loss of function
Autosomal recessive
Bailey et al.60
Luteinizing hormone receptor
Familial hypogonadism
Leydig cell hypoplasia (males)
Primary amenorrhea (females)
Simple loss of function
Autosomal recessive
Themmen &
Huhtaniemi34
Follicle stimulating hormone
receptor
Sperm-related hypofertility (males)
Ovarian dysgenesis (females)
Simple loss of function
Autosomal recessive
Themmen &
Huhtaniemi34
Gonadotropin-releasing
hormone receptor
Central hypogonadotropic hypogonadism
Simple loss of function
Autosomal recessive
Bédécarrats &
Kaiser37
KiSS 1 receptor
Central hypogonadotropic hypogonadism
Simple loss of function
Autosomal recessive
de Roux40
NK3R (TACR3)
Central hypogonadotropic hypogonadism
Simple loss of function
Autosomal recessive
Tapaloglu et al.102
Prokineticin receptor 2
Central hypogonadotropic hypogonadism
and anosmia (Kallmann syndrome)
Unknown
Autosomal recessive*
Codominant*
Digenic*
Sarfati et al.48
Relaxin receptor
Cryptorchidism in mice
Unknown in humans
Simple loss of function
Unknown
Unknown
Feng et al.103
Thyrotropin-releasing hormone
receptor
Central hypothyroidism
Simple loss of function
Autosomal recessive
Collu et al.38
TSH receptor
Euthyroid hyperthyrotropinemia
Congenital hypothyroidism
Simple loss of function
Autosomal dominant
Autosomal recessive
Vassart104
Growth-hormone-releasing
hormone
Short stature (growth hormone deficiency)
Simple loss of function
Autosomal recessive
Martari &
Salvatori36
Ghrelin receptor
Short stature
Loss of basal activity
Dominant*
Pantel et al.52
Melanocortin 4 receptor
Extreme obesity
Loss of basal activity
Codominant
Srinivasan et al.49
Parathyroid hormone and
parathyroid related protein
Bloomstrand chondrodysplasia
Simple loss of function
Autosomal recessive
Thakker et al.105
Calcium-sensing receptor
Benign familial hypocalciuric hypercalcemia
Neonatal severe primary hyperparathyroidism
Simple loss of function
Autosomal dominant
Autosomal recessive
Riccardi & Brown45
All receptors are members of subfamily A apart from parathyroid hormone receptor and parathyroid related protein receptor (subfamily B), growth-hormone-releasing hormone receptor
(subfamily B) and calcium sensor (subfamily C). *Mode of inheritance is uncertain.
the residual activity of the alleles present in individual
homozygous or compound heterozygous patients.
Loss-of-function mutations of GPCRs have a diverse
range of mechanistic consequences (Figure 2). As we now
know that GPCRs can exist as dimers or oligomers, one
might expect to observe dominant-negative effects in
some heterozygous individuals. Such effects have been
described in obese patients with mutations in the gene
that encodes melanocortin 4 receptor (MC4R)27,28 and,
outside the endocrine field, in forms of retinitis pigmentosa caused by mutations in the gene that encodes rhodopsin.29 This dominant-negative effect has been related
to defective routing of the complex formed between the
wild type and mutated receptors to the plasma membrane.30,31 Nevertheless, with a few exceptions,32 expression of the disease in heterozygous individuals is usually
mild or absent. For instance, TSH levels are only marginally and sporadically raised in heterozygote patients who
have a null allele of the TSH receptor,33 and heterozygotes
for null follicle-stimulating hormone (FSH), luteinizing
hormone, growth-hormone-releasing hormone (GHRH),
gonadotropin-releasing hormone (GnRH), thyrotropin­releasing hormone (TRH) or KiSS 1 receptor (also
known as GPR54) mutants are asymptomatic.34–40 Data
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from the past 5 years on the activation mechanisms in
homodimeric or heterodimeric receptors might provide
an explanation for this observation: activation would
induce a functional asymmetry in the dimers, with only
one protomer activated.41–43 Loss of function of one of the
protomers (by mutation or interaction with an inverse
agonist) might even favor activation of the G protein by
the dimer.44
The dominant transmission of benign familial hypo­
calci­u ric hypercalcemia, caused by mutation of the
calcium sensor, might be explained by the unusual inverse
effect of activation of this receptor on target-­tissue function (parathyroid hormone secretion).45 In this particular instance, haploinsufficiency of the calcium sensor
results in a gain of function—inappropriate secretion
of parathyroid hormone. Homozygote mutations are
responsible for the much more severe neonate hyperpara­
thyroidism phenotype than that seen in benign familial
hypocalciuric hypercalcemia.
Homozygous mutations in the gene that encodes prokineticin receptor 2 (PROKR2) causes hypogonadotropic
hypogonadism associated with anosmia (Kallmann syndrome). However, the mode of genetic transmission is
still unclear, as putative heterozygotes (a single mutation
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a
βarr
αs
β γ
αs
β γ
αθ
β γ
αθ
β γ
MAPK
SRC
Other
βarr
MAPK
SRC
Other
αs
αθ
β γ ? β γ
b
αs
β γ
αθ
β γ
Response
βarr
MAPK
SRC
Other
Loss of basal activity
c
Mutant
0–
0
βarr
αs
αθ
β γ ? β γ
WT
Log[agonist]
MAPK
SRC
Other
d
βarr
MAPK
SRC
Other
ER
Constitutively desensitized
e
f
Grossly denatured or truncated
g
h
i
ER
3
αs
β γ
Intracelluar
retention
αθ
β γ
Loss of
agonist binding
αs
β γ
Loss of
intramolecular
activation
αθ
β γ
Loss of binding
of G proteins
αs
β γ
1
αθ
β γ
2
βarr
MAPK
Loss of interaction
SRC
Other
with one G protein,
βarr, or interacting proteins
Figure 2 | GPCR loss-of-function mutations. a | GPCRs are synthesized in the RER. An interacting protein (green) is needed to
route some GPCRs, such as MC2R, to the plasma membrane; GPCRs might be silent (blue), or display basal activity (pink).
The N‑terminus of MC2R is a tethered partial agonist of the G protein. Agonist (yellow) binding activates G protein-dependent
and β arrestin-dependent effects. Desensitization of the receptor by internalization maintains activation of β arrestindependent effects. G protein-dependent effects might also continue.100,101 b | Specific mutations affect only basal activity.
c | Some mutations cause constitutive desensitization. d | Classic loss-of-function mutations affect gross protein structure,
trapping the receptor in the RER. e | Some mutations affect the interacting protein function required to route some GPCRs to
the plasma membrane. f | Other mutations interfere with agonist binding, g | with the intramolecular conformational change
involved in activation, h | or with the ability to bind G proteins. i | Mutations affecting interaction of GPCRs with one G protein,
when the receptor is coupled to multiple G proteins (1), βarr (2) or interacting proteins (3) cause biased activation.
Abbreviations: βarr, β arrestin; GPCRs, G protein-coupled receptors; MAPK, mitogen-activated protein kinase; MC2R,
melanocortin 2 receptor; RER, rough endoplasmic reticulum; SRC, proto-oncogene tyrosine-protein kinase Src.
identified) are symptomatic. Of interest, some patients
with Kallmann syndrome who have a single PROKR2
mutation also have a mutated KAL1 allele. This observation suggests that in these patients, Kallmann syndrome
might have a digenic pattern of inheritance.46–48
Basal activity
The physiological meaning of the constitutive acti­
vity displayed by some GPCRs when tested in vitro was
uncertain until natural mutants of MC4R and the growth
hormone secretagogue receptor (GHSR; also known as
NATURE REVIEWS | ENDOCRINOLOGY VOLUME 7 | JUNE 2011 | 365
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ghrelin receptor) were identified that specifically affected
their basal activity. Classic loss-of-function mutations
of the MC4R (those affecting response to α melanocyte­stimulating hormone [αMSH]) have been identified in
severely obese patients.49 As expected (there are hundreds
of ways to destroy a functional structure), these mutations
involve amino acid residues located all over the primary
structure of the protein.49 However, a class of mutations
located in the N‑terminal domain were identified whose
effects were to decrease the basal activity of the mutant
receptors.50 These mutations did not affect the potency
of αMSH or the inverse agonist agouti-related protein on
the response of receptors transiently expressed in human
embryonic kidney (HEK) 293 cells. However, an effect of
the mutations on the efficacy of αMSH has not been definitively excluded. In addition to being the first to indicate that
basal activity of GPCRs has a functional role, these observations led to the identification of the N‑terminus of MC4R
as a built-in tethered partial agonist of this receptor.50
GHSR binds ghrelin to positively control appetite and
food intake; this receptor provides a slightly different
example of the role of basal GPCR activity. To date, only
one null allele of GHSR has been identified in a compound
heterozygote male patient (aged 6.5 years) with short
stature.51 His second GHSR mutated allele had partially
lost basal activity while responding normally to ghrelin.
Besides, two families had been identified in which short
stature is segregated with a GHSR mutation (Ala204Glu)
that is characterized by substantially decreased basal
activity of the receptor.52 When the mutant GHSR was
expressed in HEK293 cells at the same level as the wildtype receptor, ghrelin stimulated it with normal potency
and efficacy.52 However, it is possible that the Ala204Glu
mutant of GHSR would show decreased expression in the
patients in vivo.53 Of the individuals who were identified
as heterozygous for this mutation, not all had short stature.
This observation is compatible with codominant trans­
mission of the trait, with incomplete penetrance of the
phenotype.52 The Ala204Glu mutation, and another with
a similar functional characteristic (Phe279Leu), might also
be associated with puberty-onset obesity.54
Considering the codominant mode of transmission
of the associated phenotypes, these observations suggest
that the physiological mechanisms controlled by MC4R
and GHSR would be highly sensitive to the level of their
basal activity. Given the large number of GPCRs that
display basal activity, extension of such observations to
more patients and additional diseases is likely; however,
the challenge will be identification of the corresponding
phenotypes.
To our knowledge, naturally occurring mutations that
affect GPCR sensitivity without modification of basal acti­
vity have not been reported. However, there is no reason to
exclude this possibility, as a mutation of this kind has been
engineered experimentally in the TSH receptor in vitro.55
Mutations and biased agonism
Many artificial mutations have been engineered (in vitro
and in animal models) that modify the coupling of GPCRs
to various G proteins or that modify β arrestin-­dependent
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effects,22,56 but there are still only a few cases in which
natural mutations have been shown to do the same.57 In
one such case, a TSH receptor mutant retained sub­normal
response for cAMP signaling, while completely losing
the ability to activate inositolphosphate generation.57 The
associated phenotype was an imbalance between iodide
trapping (cAMP-dependent) and thyroid hormone synthesis (dependent on inositolphosphate and calcium).
Natural GPCR mutations with biased agonistic effects
on G protein-dependent versus β arrestin-dependent
mechanisms will probably be identified in the future.
Here again, one difficulty will doubtless be to define the
expected phenotypes.
Mutations and interacting proteins
Receptor activity modifying proteins (RAMPs), that
interact with the calcitonin receptor and the calcitoninreceptor-like receptor, were the first to draw attention to
the ability of interacting proteins to route some GPCRs
to the plasma membrane and to modify their speci­fi­
city.58 Although natural variations in RAMPs have been
described,59 no naturally occurring mutation that causes
disease has been identified, either in RAMPs themselves
or in their cognate GPCRs.
In families presenting with glucocorticoid deficiency
type 2 (that is, those in which a mutation in the adrenocorticotropin receptor [MC2R] has been excluded60), a
search for the genetic cause of the disease led to the identification of MC2R accessory protein (MRAP),61,62 which
is required to route the MC2R to the plasma membrane.
Naturally occurring mutations of MRAP provide the first
example of a disease caused by mutations that interfere
with the binding or function of GPCR accessory proteins.
Although not in the endocrine field, mutation of the
C‑terminal tail of the metabotropic glutamate receptor 7
(MGLUR7) in mice abolishes interaction of the receptor
with Pick1 (protein interacting with C kinase 1), causing
an absence epilepsy phenotype (mice experience recurrent
loss of consciousness and an electroencephalogram shows
generalized spike-and-wave discharges).63
As there are now many proteins that are known to interact with GPCRs and to modify their function,23,24,64,65 it is
expected that many additional phenotypes will be identified secondary to the loss of such interactions. As the likely
phenotypes will probably be different from simple loss of
function of the receptors, here also the difficulty will be to
recognize them.
Gain-of-function mutations
Theoretically, gain of function might have several meanings for a hormone receptor (Figures 3 and 4). For example,
gain-of-function mutations can cause activation in the
absence of a ligand (constitutive activity); increased sensi­
tivity to the receptor’s usual agonist; increased or de novo
sensitivity to an allosteric modulator; or broadening of its
specificity. Receptors are frequently part of a chemostat, in
which case activation in the absence of a ligand is expected
to cause tissue autonomy, whereas increased sensitivity to
the receptor’s usual agonist would adjust the agonist concentration to a lower value. Following increased sensitivity
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αs
β γ
αq
β γ
αs
β γ
αq
β γ
MAPK
SRC
Other
Response
βarr
Response
a
0–
0
b
0–
Expression
0
Log (agonist)
Increased sensitivity to the agonist
αs
β γ
αq
β γ
MAPK
SRC
Other
Log (agonist)
βarr
αs
β γ
αq
β γ
Response
βarr
MAPK
SRC
Other
Increased sensitivity to the normal agonist
in the presence of an allosteric modulator
Figure 3 | Gain-of-function mutations of GPCRs. Wild-type, silent is shown in blue, wild-type with basal activity in yellow,
mutated with increased basal activity in orange and mutated, almost totally activated and nonresponsive in red. a | Wild-type
(blue and yellow) or mutated (red with a yellow dot) GPCRs might display very different levels of constitutive activity and
response to their normal agonist. The curves to the right illustrate the basal activity and responses of wild-type GPCRs
(totally silent, or with basal activity) and two examples of mutants with increasing constitutive activity, red and orange
curves). b | Top, mutations might cause increased sensitivity to the normal agonist with minimal change in basal activity (as
in some calcium sensor gain-of-function mutations, or in the case of increased amounts of receptors at the cell surface).
Conceivably, other mutations (bottom) might render a GPCR sensitive to a normally inert positive allosteric modulator, also
resulting in an increase in sensitivity to the normal agonist. Abbreviations: βarr, β arrestin; GPCRs, G protein-coupled
receptors; MAPK, mitogen-activated protein kinase; SRC, proto-oncogene tyrosine-protein kinase Src.
to an allosteric modulator or broadening of the receptor’s
specificity to a nonphysiological agonist, inappropriate
stimulation of the target will occur because the illegitimate agonist or modulator are not expected to be subject
to the normal negative feedback mechanisms. If a gainof-function mutation that results in GPCR activation in
the absence of a ligand occurs in a single cell that normally expresses the receptor (somatic mutation), the cell
will become symptomatic only if the regulatory cascade
controlled by the receptor is mitogenic in this particular
cell type or, during development, if the mutation affects
a progenitor cell that makes a substantial contribution to
the final organ. Autonomous activity of the receptor will
cause clonal expansion of the mutated cell. If the regulatory cascade also positively controls function, the resulting tumor might progressively take over function of the
normal tissue and ultimately result in autonomous hyperfunction. If the mutation is present in all cells of an organism (germ line mutation) autonomy will be displayed by
the whole organ. Gain-of-function mutations affecting
GPCRs cause a range of endocrine diseases; examples
include Bartter syndrome type V and non­autoimmune
familial hyperthyroidism (Table 2).
Germ line activating mutations
As expected, germ line activating mutations are transmitted in an autosomal or X‑linked dominant mode. In
the nephro­genic syndrome of inappropriate anti­diuresis
(NSIAD, secondary to activating mutations of the X‑linked
AVPR2 receptor 66), expression of the disease in women
might be subject to variation that is attributable to the
possibility of skewed inactivation of the X chromosome
(preferential rather than random inactivation of a given
X chromosome).67 For GPCRs such as the TSH, luteinizing hormone or choriogonadotropin receptors, which are
capable of activating more than one G protein-­dependent
cascade (Gs and Gq), the question arises whether mutations with a different effect on the two cascades would
be associated with different phenotypes. Studies in mice68
and a report in humans69 suggest that activation of Gq
might be required to observe development of goiters in
patients with nonautoimmune familial hyperthyroidism.
However, when tested in transfected nonthyroid cells, all
identified gain-of-function mutations of the TSH receptor
constitutively stimulate Gs, with only a minority capable
of stimulating both Gs and Gq.70,71 In addition, thyroid
adenomas or multinodular goiter are frequent in patients
with McCune–Albright syndrome, which is characterized by pure Gs stimulation.72 The situation is different
for the luteinizing hormone/chorio­gonadotropin receptor. All activating mutations of this GPCR cause malelimited precocious puberty and constitutively activate
Gs-dependent cAMP accumulation.34,73 Only one particular amino acid substitution, Asp478His, also causes
Leydig-cell adenoma.74 This particularity, which might be
coined ‘biased gain of function’, has been associated with
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α β
α β
FSH
hCG
Response
a
WT FSH-R
FSH
hCG
0–
0
Silent
α β
α β
FSH
hCG
Ectodomain
mutant
Response
b
FSH
hCG
c
α β
α β
FSH
Transmembrane
mutant
hCG
Response
0–
0
Silent
0–
0
Basal activity
FSH
hCG
Log (agonist)
Figure 4 | Gain-of-function mutations that affect GPCR specificity: the example of the glycoprotein hormone receptors. Wildtype silent receptor is shown in blue; position of the mutation is indicated as a yellow dot; basal activity, when present, is
shown in pink and agonist activated receptor in red. a | The wild-type FSH receptor is totally silent and activated only by FSH
and not by hCG. b | Rare mutations of the ectodomain increase slightly recognition of hCG by the FSH receptor, while keeping
the receptor silent and with a normal response to FSH. This modest gain of function is enough to cause disease
(spontaneous ovarian hyperstimulation syndrome) because of the very high concentration of hCG during pregnancy.
c | Mutations causing partial unlocking of the GPCR domain of the receptor trigger some basal activity and render the mutant
abnormally sensitive to hCG. Here again, the gain of function is modest, but enough to cause disease during pregnancy.
Abbreviations: FSH, follicle-stimulating hormone; GPCR, G protein-coupled receptor; hCG, human chorionic gonadotropin.
the ability of Asp478His to activate phospho­lipase C via
coupling to Gq, in addition to Gs.74
The FSH receptor
The FSH receptor is peculiar in humans as it is totally silent
in the absence of a ligand and not activated by many of the
amino acid substitutions that cause constitutive activity
of its close homologs, the TSH and luteinizing hormone/
choriogonadotropin receptors.75 This characteristic has
been associated with the necessity of humans to avoid
promiscuous activation of the FSH receptor by chorionic
gonadotropin (CG) during pregnancy (see below).76 A
simple gain-of-function phenotype has been observed in
just one male patient with a Asp567Gly mutation of the
FSH receptor presenting with sustained spermato­genesis
despite previous hypophysectomy.77
The KiSS 1 receptor
The KiSS 1 receptor has a key role in the development of
human puberty.40 As loss-of-function mutations of KiSS 1
cause autosomal recessive or sporadic hypo­gonadotropic
hypogonadism, it was logical to assume that gain of function of the same receptor would cause precocious puberty.
To date, a heterozygous mutation in KiSS 1 has been identified in one girl with idiopathic precocious puberty.78 The
mutation affects the C‑terminal segment of the receptor
(Arg386Pro) and, interestingly, its sole functional anomaly
is delayed desensitization of the receptor.78
368 | JUNE 2011 | VOLUME 7
AVPR2
Mutations with expected gain-of-function effects on receptor structure might unexpectedly cause a loss-of-function
phenotype. One well-studied example is provided by
mutations that affect residue 137 of the AVPR2 receptor.
Arg137His is one of the many mutations in AVPR2 that
causes nephrogenic diabetes insipidus.79 This mutation
affects one of the most conserved residues in rhodospinlike GPCRs (the R of the canonical DRY/W motif implicated in activation of class A GPCRs). Unexpectedly, two
other mutations of the same residue, Arg137Cys and
Arg137Leu, were identified as gain-of-function mutations
that cause NSIAD.66 All three mutants of AVPR2 display
some characteristics associated with gain of function: they
spontaneously recruit β arrestin and are partially constitutively desensitized. In addition, they lose the capacity
to be activated by vasopressin for cAMP generation and
activation of mitogen-activated protein kinase (MAPK,
also known as ERK) 1 or MAPK3, but still respond to the
hormone for internalization. The only difference lies in
the inability of Arg137His to stimulate basal cAMP production.80 This example illustrates how unclear the distinction might be between loss of function and gain of function
GPCR mutations, depending on the criterion used (for
example, change in conformation, recruitment of accessory
proteins, coupling to G proteins or disease phenotype).
Of a simpler nature, and already discussed for lossof-function mutations, the peculiarities of parathyroid
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Table 2 | Gain-of-function mutations of GPCRs causing endocrine diseases
Receptor
Disease
Mechanism
Mode of inheritance
References
Arginine vasopressin receptor 2
Nephrogenic syndrome of inappropriate
antidiuresis
Increased constitutive activity
X-linked dominant
Feldman
et al.66
Luteinizing hormone receptor
Male-limited precocious puberty
Increased constitutive activity
Autosomal dominant
Shenker73
Follicle stimulating hormone
receptor
In females: spontaneous ovarian
hyperstimulation syndrome
Broadening of specificity
and constitutivity
Autosomal dominant
Smits et al.90
Vasseur et al.91
TSH receptor
Nonautoimmune familial hyperthyroidism
Familial pregnancy-limited hyperthyroidism
Increased constitutive activity
Broadening of specificity
Autosomal dominant
One dominant germ
line family described
Vassart104
Rodien et al.89
KiSS 1 receptor
Precocious puberty
Decreased desensitization
One isolated case
described
Teles et al.78
Parathyroid hormone and
parathyroid related protein receptors
Jansen metaphyseal chondrodysplasia
Increased constitutive activity
Autosomal dominant
Thakker
et al.105
Calcium-sensing receptor
Familial hypocalcemic hypercalciuria
(autosomal dominant hypoparathyroidism)
Bartter syndrome type V
Increased sensitivity to calcium
Autosomal dominant
Fully activated at physiological
calcium concentrations
Autosomal dominant
Riccardi
& Brown45
Vargas-Poussou
et al.84
Associated with type 2 diabetes mellitus
Increased expression in islets
controls negatively insulin secretion
Associated with type 2
diabetes mellitus
Rosengren
et al.82
Luteinizing hormone receptor
Leydig cell adenomas with precocious
puberty
Increased constitutive activity
NA
Shenker73
TSH receptor
Autonomous thyroid adenomas
(rare carcinomas)
Increased constitutive activity
NA
Vassart104
Germ line mutations
α2A-Adrenergic receptor
Somatic mutations
All receptors are members of subfamily A apart from parathyroid hormone receptor and parathyroid-related protein receptor (subfamily B) and the calcium-sensing receptor (subfamily C).
Abbreviations: GPCRs, G protein-coupled receptors; NA, not applicable.
hormone regulation mean that gain-of-function mutations of the calcium sensor cause hypofunction of the
parathyroid gland.
Increased GPCR expression
For GPCRs with low or absent basal activity, increased
receptor expression is expected to increase the sensiti­
vity of the tissue to the agonist, hence to lower the steady
state concentration of the agonist, if the agonist–receptor
couple is part of a well-controlled chemostat. Differences
in the strength of individual GPCR alleles might thus contribute to the distribution of normal circulating agonist
concentrations in the population.
An interesting observation has been made in an example
where the GPCR involved is not implicated in a simple
chemostat. The α2A-adrenergic receptor (ADRA2) is
known, among many other roles, to control insulin secretion.81 In congenic strains of the diabetic Goto-Kakizaki
rat, an Adra2 allele has been identified that is associated
with overexpression of the receptor and reduced insulin
production by isolated islets of Langerhans.82 This discovery led to identification in a cohort of patients with
diabetes mellitus of a homo­logous human ADRA2 riskallele, and demonstration that the variant receptor causes
a similar in vitro pheno­type when tested in human islets
of Langerhans.82 However, it must be stressed that contrary to disease-causing mutants, this ADRA2 allele is
present in the human population as a relatively frequent
poly­morphism (allele frequency ~15%),82 which is simply
statistically associated with type 2 diabetes mellitus.
Increased sensitivity to modulators
The allosteric model for GPCR activation predicts that
constitutively active mutants would display increased
sensitivity to their normal agonists.21 Mutations of the
calcium-sensing receptor provide a nice illustration
of this phenomenon: in familial hypocalcemic hyper­
calciuria (quite a benign condition) the phenotype results
from an increased sensitivity of the mutant receptor to
plasma levels of calcium, rather than from its basal acti­
vity.83 In the more severe Bartter syndrome type V, the
receptor is almost fully active even when exposed to very
low concentrations of calcium.84
In other diseases, the contribution that increased
sensitivity of the mutant receptor to its normal agonist
makes to the phenotype is difficult to appreciate, as it is
dominated by the hormone-independent effects of the
mutations and obliterated by the negative feedback.
Outside human endocrinology, an observation from
a study published in 2010 is worth mentioning as it is
compatible with the existence of mutations unmasking
stimulation by allosteric modulators. In a genome-wide
search for mutations associated with domestication of
the chicken, Rubin et al. identified a single amino acid
substitution in the TSH receptor as the strongest candidate.85 TSH receptor expressed in ependymal cells has a
key role in the adaptation of birds and mammals to the
length of the day.86,87 As a consequence, it is tempting to
hypothesize that domestication has selected an allele of
the TSH receptor with increased sensitivity to an allo­
steric modulator, or agonist, which would be present only
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REVIEWS
in the cerebrospinal fluid, thus leading to the laying of
eggs all year round.
Widening of receptor specificity
The degree of specificity of wild-type GPCRs is extremely
variable, going from complete specificity for most
hormone receptors, to promiscuous activation by a wide
spectrum of agonists for chemokine receptors. Naturally
occurring mutations that cause promiscuous GPCR activation have only been described in the genes that encode
the TSH and FSH receptors. In both cases, the same
illegi­timate agonist is involved—human CG (hCG). The
β subunit of hCG is a close paralog of TSHβ and FSHβ, and
a recent evolutionary addition in the genome of simians.88
In higher primates, intact CG (a heterodimer of the
α and β subunits, known as the holohormone) reaches
plasma concentrations several orders of magnitude higher
than TSH or FSH during pregnancy. Hence, even receptor
mutations with a very mild gain of function in response to
hCG will become symptomatic during pregnancy.
A single family has been described in which pregnancylimited hyperthyroidism segregates as an autosomal
dominant trait.89 The causal mutation is in the hormonerecognition domain of the TSH receptor, making it
respond to hCG inappropriately.89 Of interest, in addition to hyperthyroidism, the affected women in this
family suffered severe hyperemesis gravidarum during
each pregnancy and experienced abortion if they weren’t
treated with antithyroid drugs. This finding suggests that
illegitimate activation of extrathyroidal TSH receptor
might be part of the syndrome. One explanation of the
extreme rarity of this disease could be underdiagnosis. In
many patients, spontaneous abortion might be the initial
manifestation, before hyperthyroidism is diagnosed.
The situation is different for the FSH receptor. Several
families and individual patients have been identified in
which a mutated FSH receptor is associated with spontaneous ovarian hyperstimulation syndrome (OHSS)
during early pregnancy.90–92 In each case, the mutant
FSH receptor displayed abnormal sensitivity to hCG.
Interestingly, among the patients identified to date, only
one has a mutation that affects the hormone-recognition domain of the receptor.93 The other patients carried
FSH receptors with a typical gain-of-function mutation
that affected residues implicated in the silencing locks
of GPCRs. Accordingly, these mutants displayed low,
but detectable, constitutive activity. In addition to their
increased response to hCG, they also responded abnormally to TSH, which suggested that similar mutations
might be found in patients with severe hypothyroidism
who might also develop OHSS. To date, no such mutation has been identified in these patients, suggesting
that OHSS in women with hypo­thyroidism is attributable to the low, but detectable, sensitivity of the wild-type
receptor to TSH.94 These observations establish a link
between basal activity of the FSH receptor and its functional specificity. They suggest that partial activation of
a receptor that is usually totally silent makes it prone to
activation by low-affinity agonists (hCG or TSH). From
an evolutionary point of view, it seems that the TSH and
370 | JUNE 2011 | VOLUME 7
FSH receptors have selected different strat­egies to avoid
promiscuous activation by hCG in humans: the former
using the selectivity of its hormone-binding domain,95
while the latter increased negative constraint in the
absence of an agonist.76
Somatic mutations
Somatic gain-of-function mutations of GPCRs have been
identified in two endocrine organs: the thyroid gland
and the testes (Table 2). In the thyroid gland, activating
mutations of the TSH receptor are the primary cause of
autonomous thyroid adenomas (50–80%), the second
being mutation of Gsα.71 The frequent occurrence of the
disease in Europe enabled identification of the majority
of the residues in which mutation would cause constitutive activation. The structure–function relationships of
the mutant receptors have been reviewed in the light
of structural data.76 No relation could be made between
coupling of the receptor (to Gs only or to both Gs and Gq)
and the phenotype of the tumors.71 In the rare thyroid
carcinomas harboring an activated TSH receptor mutant
there is no convincing bias between Gs and Gq coupling
of the mutants.96,97
In the testes, out of the panel of activating mutations
that cause male-limited precocious puberty and Leydig
cell hyperplasia, only one (Asp578His) is a somatic mutation and causes Leydig cell adenoma in young boys with
precocious puberty.73,98 Of interest, other amino acid substitutions at residue 578 are frequent causes of the germ
line-dependent phenotype but never found in Leydig cell
tumors. The observation that Asp578His is a very strong
allele capable of activating both Gs and Gq provides a
tempting explanation to its tumorigenic potential.
Conclusions
During the past 20 years, the study of structure–function
relationships of natural GPCR mutants has contributed
considerably to our understanding of how these important
sensors function in vivo. The list of phenotypes associated
with mutations that affect the canonical G protein-related
effects of GPCRs must be close to completion. For the
additional G protein-independent regulatory mechanisms described briefly in this Review, we are only at the
start of the road. Despite the great power of mouse or rat99
mutagenesis and transgenenesis to unravel phenotype–­
genotype relationships, it is expected that the study of
human genetic traits or diseases will remain fruitful. We
hope that the present Review will whet the appetite of
the clinical endocrinologist reader to identify novel and
interesting cases from among his or her patients.
Review criteria
PubMed was searched using the terms “GPCR and
mutation”, “GPCR and loss of function”, “GPCR and gain
of function”. Original articles, reviews, editorials and their
reference lists were considered. Articles published between
1989 and 2010 were included. The 2010 (6th) edition
of Endocrinology (eds Jameson, J. L. & De Groot, L. J.,
Saunders, Philadelphia, 2010) was also searched.
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Author contributions
Both authors contributed equally to all aspects of
this Review.
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