REVIEWS 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 www.nature.com/nrendo

© 2011 Macmillan Publishers Limited. All rights reserved

REVIEWS 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

NATURE REVIEWS | ENDOCRINOLOGY

VOLUME 7  |  JUNE 2011  |  363 © 2011 Macmillan Publishers Limited. All rights reserved

REVIEWS 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 364  |  JUNE 2011  |  VOLUME 7



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 www.nature.com/nrendo

© 2011 Macmillan Publishers Limited. All rights reserved

REVIEWS 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 © 2011 Macmillan Publishers Limited. All rights reserved

REVIEWS 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 366  |  JUNE 2011  |  VOLUME 7



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 www.nature.com/nrendo

© 2011 Macmillan Publishers Limited. All rights reserved

REVIEWS

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

NATURE REVIEWS | ENDOCRINOLOGY

VOLUME 7  |  JUNE 2011  |  367 © 2011 Macmillan Publishers Limited. All rights reserved

REVIEWS

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 www.nature.com/nrendo

© 2011 Macmillan Publishers Limited. All rights reserved

REVIEWS 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

NATURE REVIEWS | ENDOCRINOLOGY

VOLUME 7  |  JUNE 2011  |  369 © 2011 Macmillan Publishers Limited. All rights reserved

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.

www.nature.com/nrendo © 2011 Macmillan Publishers Limited. All rights reserved

REVIEWS 1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14. 15. 16.

17.

18.

19.

20.

Hirai, T., Subramaniam, S. & Lanyi, J. K. Structural snapshots of conformational changes in a seven-helix membrane protein: lessons from bacteriorhodopsin. Curr. Opin. Struct. Biol. 19, 433–439 (2009). Fredriksson, R., Lagerström, M. C., Lundin, L. G. & Schiöth, H. B. The G‑protein‑coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol. Pharmacol. 63, 1256–1272 (2003). Lania, A. G., Mantovani, G. & Spada, A. Mechanisms of disease: Mutations of G proteins and G‑protein‑coupled receptors in endocrine diseases. Nat. Clin. Pract. Endocrinol. Metab. 2, 681–693 (2006). Schöneberg, T. et al. Mutant G‑protein‑coupled receptors as a cause of human diseases. Pharmacol. Ther. 104, 173–206 (2004). Thompson, M. D., Percy, M. E., McIntyre Burnham, W. & Cole, D. E. G protein-coupled receptors disrupted in human genetic disease. Methods Mol. Biol. 448, 109–137 (2008). Spiegel, A. M. Inherited endocrine diseases involving G proteins and G protein-coupled receptors. Endocr. Dev. 11, 133–144 (2007). Lefkowitz, R. J. Historical review: a brief history and personal retrospective of seventransmembrane receptors. Trends Pharmacol. Sci 25, 413–422 (2004). Lefkowitz, R. J. Seven transmembrane receptors: something old, something new. Acta Physiol. (Oxf) 190, 9–19 (2007). Palczewski, K. et al. Crystal structure of rhodopsin: A G protein-coupled receptor. Science 289, 739–745 (2000). Rosenbaum, D. M., Rasmussen, S. G. & Kobilka, B. K. The structure and function of G‑protein‑coupled receptors. Nature 459, 356–363 (2009). Jaakola, V. P. et al. The 2.6 angstrom crystal structure of a human A2A adenosine receptor bound to an antagonist. Science 322, 1211–1217 (2008). Tate, C. G. & Schertler, G. F. Engineering G protein-coupled receptors to facilitate their structure determination. Curr. Opin. Struct. Biol. 19, 386–395 (2009). GPCRDB information system for G proteincoupled receptors [online], http:// www.gpcr.org/7tm/ (2010). GPCR natural variants database [online], http://nava.liacs.nl/ (2007). TinyGRAP mutant database [online], http:// www.cmbi.ru.nl/tinygrap/credits/ (2010). Cotecchia, S., Exum, S., Caron, M. G. & Lefkowitz, R. J. Regions of the α1‑adrenergic receptor involved in coupling to phosphatidylinositol hydrolysis and enhanced sensitivity of biological function. Proc. Natl Acad. Sci. USA 87, 2896–2900 (1990). Kjelsberg, M. A., Cotecchia, S., Ostrowski, J., Caron, M. G. & Lefkowitz, R. J. Constitutive activation of the α1B-adrenergic receptor by all amino acid substitutions at a single site. Evidence for a region which constrains receptor activation. J. Biol. Chem. 267, 1430–1433 (1992). Hofmann, K. P. et al. A G protein-coupled receptor at work: the rhodopsin model. Trends Biochem. Sci. 34, 540–552 (2009). Scheerer, P. et al. Crystal structure of opsin in its G‑protein‑interacting conformation. Nature 455, 497–502 (2008). Rasmussen, S. G. et al. Structure of a nanobody-stabilized active state of the β2 adrenoceptor. Nature 469, 175–180 (2011).

21. Samama, P., Cotecchia, S., Costa, T. & Lefkowitz, R. J. A mutation-induced activated state of the β2‑adrenergic receptor. Extending the ternary complex model. J. Biol. Chem. 268, 4625–4636 (1993). 22. Rajagopal, S., Rajagopal, K. & Lefkowitz, R. J. Teaching old receptors new tricks: biasing seventransmembrane receptors. Nat. Rev. Drug Discov. 9, 373–386 (2010). 23. Bockaert, J., Perroy, J., Bécamel, C., Marin, P. & Fagni, L. GPCR interacting proteins (GIPs) in the nervous system: roles in physiology and pathologies. Annu. Rev. Pharmacol. Toxicol. 50, 89–109 (2010). 24. Ritter, S. L. & Hall, R. A. Fine-tuning of GPCR activity by receptor-interacting proteins. Nat. Rev. Mol. Cell Biol. 10, 819–830 (2009). 25. Lohse, M. J. Dimerization in GPCR mobility and signaling. Curr. Opin. Pharmacol. 10, 53–58 (2010). 26. Bouvier, M. Oligomerization of G‑protein‑coupled transmitter receptors. Nat. Rev. Neurosci. 2, 274–286 (2001). 27. Tarnow, P. et al. A heterozygous mutation in the third transmembrane domain causes a dominant-negative effect on signalling capability of the MC4R. Obes. Facts 1, 155–162 (2008). 28. Biebermann, H. et al. Autosomal-dominant mode of inheritance of a melanocortin‑4 receptor mutation in a patient with severe early-onset obesity is due to a dominant-negative effect caused by receptor dimerization. Diabetes 52, 2984–2988 (2003). 29. Mendes, H. F., van der Spuy, J., Chapple, J. P. & Cheetham, M. E. Mechanisms of cell death in rhodopsin retinitis pigmentosa: implications for therapy. Trends Mol. Med. 11, 177–185 (2005). 30. Granell, S., Mohammad, S., RamanagoudrBhojappa, R. & Baldini, G. Obesity-linked variants of melanocortin‑4 receptor are misfolded in the endoplasmic reticulum and can be rescued to the cell surface by a chemical chaperone. Mol. Endocrinol. 24, 1805–1821 (2010). 31. Lubrano-Berthelier, C. et al. Melanocortin 4 receptor mutations in a large cohort of severely obese adults: prevalence, functional classification, genotype-phenotype relationship, and lack of association with binge eating. J. Clin. Endocrinol. Metab. 91, 1811–1818 (2006). 32. Calebiro, D. et al. Intracellular entrapment of wild-type TSH receptor by oligomerization with mutants linked to dominant TSH resistance. Hum. Mol. Genet. 14, 2991–3002 (2005). 33. Alberti, L. et al. Germline mutations of TSH receptor gene as cause of nonautoimmune subclinical hypothyroidism. J. Clin. Endocrinol. Metab. 87, 2549–2555 (2002). 34. Themmen, A. P. N. & Huhtaniemi, I. T. Mutations of gonadotropins and gonadotropin receptors: elucidating the physiology and pathophysiology of pituitary-gonadal function. Endocr. Rev. 21, 551–583 (2000). 35. Latronico, A. C. et al. Brief report: testicular and ovarian resistance to luteinizing hormone caused by inactivating mutations of the luteinizing hormone-receptor gene. N. Engl. J. Med. 334, 507–512 (1996). 36. Martari, M. & Salvatori, R. Chapter 3 diseases associated with growth hormone-releasing hormone receptor (GHRHR) mutations. Prog. Mol. Biol. Transl. Sci. 88, 57–84 (2009). 37. Bédécarrats, G. Y. & Kaiser, U. B. Mutations in the human gonadotropin-releasing hormone receptor: insights into receptor biology and function. Semin. Reprod. Med. 25, 368–378 (2007). 38. Collu, R. et al. A novel mechanism for isolated central hypothyroidism: inactivating mutations in

NATURE REVIEWS | ENDOCRINOLOGY

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

the thyrotropin-releasing hormone receptor gene. J. Clin. Endocrinol. Metab. 82, 1561–1565 (1997). Bonomi, M. et al. A family with complete resistance to thyrotropin-releasing hormone. N. Engl. J. Med. 360, 731–734 (2009). de Roux, N. GnRH receptor and GPR54 inactivation in isolated gonadotropic deficiency. Best Pract. Res. Clin. Endocrinol. Metab. 20, 515–528 (2006). Hlavackova, V. et al. Evidence for a single heptahelical domain being turned on upon activation of a dimeric GPCR. EMBO J. 24, 499–509 (2005). Damian, M., Martin, A., Mesnier, D., Pin, J. P. & Banères, J. L. Asymmetric conformational changes in a GPCR dimer controlled by G‑proteins. EMBO J. 25, 5693–5702 (2006). Parenty, G., Appelbe, S. & Milligan, G. CXCR2 chemokine receptor antagonism enhances DOP opioid receptor function via allosteric regulation of the CXCR2-DOP receptor heterodimer. Biochem. J. 412, 245–256 (2008). Han, Y., Moreira, I. S., Urizar, E., Weinstein, H. & Javitch, J. A. Allosteric communication between protomers of dopamine class A GPCR dimers modulates activation. Nat. Chem. Biol. 5, 688–695 (2009). Riccardi, D. & Brown, E. M. Physiology and pathophysiology of the calcium-sensing receptor in the kidney. Am. J. Physiol. Renal Physiol. 298, F485–F499 (2010). Abreu, A. P., Kaiser, U. B. & Latronico, A. C. The role of prokineticins in the pathogenesis of hypogonadotropic hypogonadism. Neuroendocrinology 91, 283–290 (2010). Dodé, C. et al. Kallmann syndrome: mutations in the genes encoding prokineticin‑2 and prokineticin receptor‑2. PLoS Genet. 2, e175 (2006). Sarfati, J. et al. A comparative phenotypic study of kallmann syndrome patients carrying monoallelic and biallelic mutations in the prokineticin 2 or prokineticin receptor 2 genes. J. Clin. Endocrinol. Metab. 95, 659–669 (2010). Tao, Y. X. The melanocortin‑4 receptor: physiology, pharmacology, and pathophysiology. Endocr. Rev. 31, 506–543 (2010). Srinivasan, S. et al. Constitutive activity of the melanocortin‑4 receptor is maintained by its N‑terminal domain and plays a role in energy homeostasis in humans. J. Clin. Invest. 114, 1158–1164 (2004). Pantel, J. et al. Recessive isolated growth hormone deficiency and mutations in the ghrelin receptor. J. Clin. Endocrinol. Metab. 94, 4334–4341 (2009). Pantel, J. et al. Loss of constitutive activity of the growth hormone secretagogue receptor in familial short stature. J. Clin. Invest. 116, 760–768 (2006). Liu, G., Fortin, J. P., Beinborn, M. & Kopin, A. S. Four missense mutations in the ghrelin receptor result in distinct pharmacological abnormalities. J. Pharmacol. Exp. Ther. 322, 1036–1043 (2007). Holst, B. & Schwartz, T. W. Ghrelin receptor mutations—too little height and too much hunger. J. Clin. Invest. 116, 637–641 (2006). Claeysen, S. et al. A conserved Asn in TM7 of the TSH receptor is a common requirement for activation by both mutations and its natural agonist. FEBS Lett. 517, 195–200 (2002). Chang, W. C. et al. Modifying ligand-induced and constitutive signaling of the human 5‑HT4 receptor. PLoS ONE 2, e1317 (2007). Grasberger, H., Van Sande, J., Hag-Dahood Mahameed, A., Tenenbaum-Rakover, Y. &

VOLUME 7  |  JUNE 2011  |  371 © 2011 Macmillan Publishers Limited. All rights reserved

REVIEWS

58.

59.

60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

70.

71.

72.

73.

74.

Refetoff, S. A familial thyrotropin (TSH) receptor mutation provides in vivo evidence that the inositol phosphates/Ca2+ cascade mediates TSH action on thyroid hormone synthesis. J. Clin. Endocrinol. Metab. 92, 2816–2820 (2007). Parameswaran, N. & Spielman, W. S. RAMPs: the past, present and future. Trends Biochem. Sci. 31, 631–638 (2006). Bailey, R. J., Bradley, J. W., Poyner, D. R., Rathbone, D. L. & Hay, D. L. Functional characterization of two human receptor activitymodifying protein 3 variants. Peptides 31, 579–584 (2010). Metherell, L. A., Chan, L. F. & Clark, A. J. The genetics of ACTH resistance syndromes. Best Pract. Res. Clin. Endocrinol. Metab. 20, 547–560 (2006). Hughes, C. R. et al. Missense mutations in the melanocortin 2 receptor accessory protein that lead to late onset familial glucocorticoid deficiency type 2. J. Clin. Endocrinol. Metab. 95, 3497–3501 (2010). Metherell, L. A. et al. Mutations in MRAP, encoding a new interacting partner of the ACTH receptor, cause familial glucocorticoid deficiency type 2. Nat. Genet. 37, 166–170 (2005). Bertaso, F. et al. PICK1 uncoupling from mGluR7a causes absence-like seizures. Nat. Neurosci. 11, 940–948 (2008). Klenk, C. et al. Formation of a ternary complex between NHERF1, β-arrestin, and parathyroid hormone receptor. J. Biol. Chem. 285, 30355–30362 (2010). Halls, M. L. & Cooper, D. M. Sub-picomolar relaxin signalling by a pre-assembled RXFP1, AKAP79, AC2, β-arrestin 2, PDE4D3 complex. EMBO J. 29, 2772–2787 (2010). Feldman, B. J. et al. Nephrogenic syndrome of inappropriate antidiuresis. N. Engl. J. Med. 352, 1884–1890 (2005). Decaux, G. et al. Nephrogenic syndrome of inappropriate antidiuresis in adults: high phenotypic variability in men and women from a large pedigree. J. Am. Soc. Nephrol. 18, 606–612 (2007). Kero, J. et al. Thyrocyte-specific Gq/G11 deficiency impairs thyroid function and prevents goiter development. J. Clin. Invest. 117, 2399–2407 (2007). Winkler, F. et al. A new phenotype of nongoitrous and nonautoimmune hyperthyroidism caused by a heterozygous thyrotropin receptor mutation in transmembrane helix 6. J. Clin. Endocrinol. Metab. 95, 3605–3610 (2010). Parma, J. et al. Diversity and prevalence of somatic mutations in the TSH receptor and Gsα genes as a cause of toxic thyroid adenomas. J. Clin. Endocrinol. Metab. 82, 2695–2701 (1997). Corvilain, B., Van Sande, J., Dumont, J. E. & Vassart, G. Somatic and germline mutations of the TSH receptor and thyroid diseases. Clin. Endocrinol. (Oxf) 55, 143–158 (2001). Chanson, P., Salenave, S. & Orcel, P. McCune‑Albright syndrome in adulthood. Pediatr. Endocrinol. Rev. 4 (Suppl. 4), 453–462 (2007). Shenker, A. Activating mutations of the lutropin choriogonadotropin receptor in precocious puberty. Receptors Channels 8, 3–18 (2002). Liu, G. et al. Leydig-cell tumors caused by an activating mutation of the gene encoding the

372  |  JUNE 2011  |  VOLUME 7



75.

76.

77.

78.

79.

80.

81.

82.

83.

84.

85.

86.

87.

88.

89.

90.

91.

luteinizing hormone receptor. N. Engl. J. Med. 341, 1731–1736 (1999). Kudo, M., Osuga, Y., Kobilka, B. K. & Hsueh, A. J. Transmembrane regions V and VI of the human luteinizing hormone receptor are required for constitutive activation by a mutation in the third intracellular loop. J. Biol. Chem. 271, 22470–22478 (1996). Vassart, G., Pardo, L. & Costagliola, S. A molecular dissection of the glycoprotein hormone receptors 3. Trends Biochem. Sci. 29, 119–126 (2004). Simoni, M., Gromoll, J. & Nieschlag, E. The follicle-stimulating hormone receptor: biochemistry, molecular biology, physiology, and pathophysiology. Endocr. Rev. 18, 739–773 (1997). Teles, M. G. et al. A GPR54-activating mutation in a patient with central precocious puberty. N. Engl. J. Med. 358, 709–715 (2008). Fujiwara, T. M. & Bichet, D. G. Molecular biology of hereditary diabetes insipidus. J. Am. Soc. Nephrol. 16, 2836–2846 (2005). Rochdi, M. D. et al. Functional characterization of vasopressin type 2 receptor substitutions (R137H/C/L) leading to nephrogenic diabetes insipidus and nephrogenic syndrome of inappropriate antidiuresis: implications for treatments. Mol. Pharmacol. 77, 836–845 (2010). Devedjian, J. C. et al. Transgenic mice overexpressing α2A-adrenoceptors in pancreatic β-cells show altered regulation of glucose homeostasis. Diabetologia 43, 899–906 (2000). Rosengren, A. H. et al. Overexpression of α2Aadrenergic receptors contributes to type 2 diabetes. Science 327, 217–220 (2010). Egbuna, O. I. & Brown, E. M. Hypercalcaemic and hypocalcaemic conditions due to calciumsensing receptor mutations. Best Pract. Res. Clin. Rheumatol. 22, 129–148 (2008). Vargas-Poussou, R. et al. Functional characterization of a calcium-sensing receptor mutation in severe autosomal dominant hypocalcemia with a Bartter-like syndrome. J. Am. Soc. Nephrol. 13, 2259–2266 (2002). Rubin, C. J. et al. Whole-genome resequencing reveals loci under selection during chicken domestication. Nature 464, 587–591 (2010). Hanon, E. A. et al. Effect of photoperiod on the thyroid-stimulating hormone neuroendocrine system in the European hamster (Cricetus cricetus). J. Neuroendocrinol. 22, 51–55 (2010). Nakao, N. et al. Thyrotrophin in the pars tuberalis triggers photoperiodic response. Nature 452, 317–322 (2008). Henke, A. & Gromoll, J. New insights into the evolution of chorionic gonadotrophin. Mol. Cell. Endocrinol. 291, 11–19 (2008). Rodien, P. et al. Familial gestational hyperthyroidism caused by a mutant thyrotropin receptor hypersensitive to human chorionic gonadotropin. N. Engl. J. Med. 339, 1823–1826 (1998). Smits, G. et al. Ovarian hyperstimulation syndrome due to a mutation in the folliclestimulating hormone receptor. N. Engl. J. Med. 349, 760–766 (2003). Vasseur, C. et al. A chorionic gonadotropinsensitive mutation in the follicle-stimulating hormone receptor as a cause of familial

gestational spontaneous ovarian hyperstimulation syndrome. N. Engl. J. Med. 349, 753–759 (2003). 92. Montanelli, L. et al. A mutation in the folliclestimulating hormone receptor as a cause of familial spontaneous ovarian hyperstimulation syndrome. J. Clin. Endocrinol. Metab. 89, 1255–1258 (2004). 93. De Leener A. et al. Identification of the first germline mutation in the extracellular domain of the follitropin receptor responsible for spontaneous ovarian hyperstimulation syndrome. Hum. Mutat. 29, 91–98 (2008). 94. De Leener A. et al. Presence and absence of follicle-stimulating hormone receptor mutations provide some insights into spontaneous ovarian hyperstimulation syndrome physiopathology. J. Clin. Endocrinol. Metab. 91, 555–562 (2006). 95. Smits, G. et al. Glycoprotein hormone receptors: determinants in leucine-rich repeats responsible for ligand specificity. EMBO J. 22, 2692–2703 (2003). 96. Russo, D. et al. Activating mutations of the TSH receptor in differentiated thyroid carcinomas. Oncogene 11, 1907–1911 (1995). 97. Spambalg, D. et al. Structural studies of the thyrotropin receptor and Gsα in human thyroid cancers: low prevalence of mutations predicts infrequent involvement in malignant transformation. J. Clin. Endocrinol. Metab. 81, 3898–3901 (1996). 98. d’Alva, C. B. et al. A single somatic activating Asp578His mutation of the luteinizing hormone receptor causes Leydig cell tumour in boys with gonadotropin-independent precocious puberty. Clin. Endocrinol. (Oxf) 65, 408–410 (2006). 99. van Boxtel R. et al. Systematic generation of in vivo G protein-coupled receptor mutants in the rat. Pharmacogenomics J. doi:10.1038/ tpj.2010.44. 100. Calebiro, D., Nikolaev, V. O., Persani, L. & Lohse, M. J. Signaling by internalized G‑protein‑coupled receptors. Trends Pharmacol. Sci. 31, 221–228 (2010). 101. Neumann, S., Geras-Raaka, E., MarcusSamuels, B. & Gershengorn, M. C. Persistent cAMP signaling by thyrotropin (TSH) receptors is not dependent on internalization. FASEB J. 24, 3992–3999 (2010). 102. Topaloglu, A. K. et al. TAC3 and TACR3 mutations in familial hypogonadotropic hypogonadism reveal a key role for Neurokinin B in the central control of reproduction. Nat. Genet. 41, 354–358 (2009). 103. Feng, S. et al. INSL3/RXFP2 signaling in testicular descent. Ann. NY Acad Sci 1160, 197–204 (2009). 104. Vassart, G. in Endocrinology (eds Jameson, J. L. & De Groot, L. J.) 1712–1720 (Saunders, Philadelphia, 2010). 105. Thakker, T. V., Bringhurst, F. R. & Jüppner, H. in Endocrinology (eds Jameson, J. L. & De Groot, L. J.) 1136–1175 (Saunders, Philadelphia, 2010). 106. Vilardaga, J.-P., Agnati, L. F., Fuxe, K. & Ciruela, F. G-protein-coupled receptor heteromer dynamics. J. Cell Sci. 123, 4215–4220 (2010). Author contributions Both authors contributed equally to all aspects of this Review.

www.nature.com/nrendo © 2011 Macmillan Publishers Limited. All rights reserved