ReviewChild Health

G Protein–Coupled Receptors in Child Development, Growth, and Maturation

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Science Signaling  12 Oct 2010:
Vol. 3, Issue 143, pp. re7
DOI: 10.1126/scisignal.3143re7

Abstract

G protein–coupled receptors (GPCRs) constitute a large family of cell membrane receptors that affect embryogenesis, development, and child physiology, and they are targets for approved drugs and those still in development. The sensitivity of GPCRs to their respective extracellular hormones, neurotransmitters, and environmental stimulants, as well as their interaction with other receptors and intracellular signaling proteins (such as receptor activity–modifying proteins), contribute to variations in child development, growth, and maturation. Here, we summarize current knowledge about the mechanisms of activation (in either the presence or absence of ligands) that lead to the sensitivities of GPCRs and their respective effects as seen throughout human developmental and maturational phases.

Introduction

Early theories of the functions of hormones and cytokines emphasized the effect of the concentration of a ligand on the physiology and pathology of child development and maturation; however, ligand signaling that occurs in the context of decreased bioavailability of cytokines or reduced sensitivity to hormones may have equally important effects on bodily processes during childhood. The effects of ligand signaling range from malfunction to enhanced function in the sphere of pathology, and from retarded to precocious in the sphere of the physiological continuum of child development. Many hormones, neurotransmitters, chemokines, local mediators, and sensory stimuli affect cells by binding to heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) (1). GPCRs, which constitute the largest superfamily of proteins, therefore regulate the function of virtually every cell, including those associated with child development, growth, and maturation. Throughout evolution, these proteins have demonstrated the capability to transduce messages in forms as varied as photons, organic odorants, nucleotides, nucleosides, peptides, lipids, and proteins (2). This superfamily of proteins is currently a major target for potential therapeutic agents (3).

GPCRs are characterized by the presence of seven membrane-spanning α-helical segments that are separated by alternating intracellular and extracellular loop regions, which are subjected to structural modifications during activation. GPCRs are capable of recruiting and regulating the activity of specific heterotrimeric G proteins, which are highly specialized transducers that are composed of three subunits: α, β, and γ. As versatile receptors, GPCRs modulate the activity of multiple signaling pathways that lead to diverse biological responses. They play an important role in homeostasis and help to maintain tight control of vital physiological processes such as cardiac function, body temperature, blood pressure, brain activity, and endocrine function (1). Therefore, it is not surprising that abnormal GPCR signaling has been identified as the underlying cause of many human diseases. Understanding the signaling pathways that are regulated by these receptors is the first step in the development of diagnostic tools and treatment for associated diseases. Here, we summarize the current knowledge of the sensitivity of GPCRs, emphasizing the importance of the activation of these membrane receptors in embryogenesis, development, and child physiology and disease.

Molecular Mechanisms of GPCR Sensitivity

GPCRs mediate most of the physiological responses to hormones. They use different domains to bind to their ligands and to activate G proteins. Their coupling to G proteins is regulated by splicing, RNA editing, and phosphorylation. Some GPCRs form homodimers or heterodimers with structurally different GPCRs or with membrane-bound proteins, such as receptor activity–modifying proteins (RAMPs). Characterization of certain GPCRs has identified a previously unknown group of membrane proteins, collectively named accessory proteins, which are important for the cell surface expression and function of GPCRs (4). These include calcitonin-like RAMP (5) and the accessory protein of the melanocortin 2 receptor (MRAP) (6). Sensitivity to GPCR ligands is multifactorial, based on the three-component unit that consists of the GPCR itself, the G protein, and G protein–regulated diverse effectors, which include adenylyl cyclase (regulated by Gs and Gi proteins), p115Rho guanine nucleotide exchange factor (GEF) (regulated by G12 family proteins), various ion channels for K+ and Ca2+ (regulated by Gβγ subunits from Gi/o proteins), phospholipase C–β (PLC-β, regulated by Gq family proteins), and cyclic guanosine monophosphate (cGMP) phosphodiesterase (regulated by Gαgust and Gαt proteins), among others. Although it was originally thought that only Gα subunits interacted with effectors, it is now clear that both guanosine triphosphate (GTP)–bound Gα subunits and Gβγ subunits regulate effector activity (1). Mechanisms exist that regulate the activities of the three major components that transduce GPCR signals, as well as those of the postreceptor cyclic adenosine monophosphate (cAMP) phosphodiesterases, phosphatidylinositol phosphatases, and diacylglycerol kinases (1). The GPCR itself is a target for inhibition by receptor desensitization, sequestration, and down-regulation. These processes result in the uncoupling of the receptor from the G protein, removal of receptors from the plasma membrane, recycling or degradation of the receptor, and reduced synthesis of new receptors. The GPCR is also a target for positive regulation. Protein interactions modulate GPCR signaling by influencing the binding of ligand to the receptor, the coupling of the GPCR to G proteins and their interactions with effectors, and the targeting of GPCRs to specific subcellular locations. The latter includes GPCR dimerization, interaction of GPCRs with RAMPs, and the binding of scaffolding proteins to the third intracellular loop and the C terminus of the receptor (1). Effector activity is modulated both by the availability of free G protein subunits and by feedback inhibition (7).

Loss-of-function mutations of GPCRs are usually associated with hormone insensitivity disorders and consequently with phenotypes that resemble complete or partial ligand deficiency (8). Distinct cellular mechanisms underlying the inactivating mutations of these receptors have been described, including decreases in the intrinsic signaling properties of the receptor, a reduction in its ligand-binding affinity, and interference in the ability of the receptor to activate G proteins (Table 1). Furthermore, a reduction in the numbers of receptors at the cell surface may also result in loss of function. These events are not mutually exclusive; a loss-of-function mutation that affects a GPCR may reduce the responsiveness of the target cell through a combination of mechanisms. In contrast, gain-of-function mutations of GPCRs are generally associated with hormonal hyperfunction disorders (9, 10). Remarkably, most of these mutations are missense alterations found in the heterozygous state, which indicate dominant inheritance. Receptor activation caused by mutations generally occurs in the absence of the ligand (exhibiting constitutive activation) and rarely in the presence of the cognate ligand or other nonspecific ligand, characterizing nonconstitutive and promiscuous receptor activation models, respectively (Table 1).

Table 1

Distinct cellular mechanisms of GPCR mutations implicated in human diseases (68).

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GPCRs can trigger signaling mechanisms that are independent of the classic G protein–mediated pathways. β-Arrestins, a small family of intracellular proteins initially identified for their role in the desensitization of GPCRs, act as signal transducers through a distinct mechanism of scaffolding with accessory effector molecules (11). For example, stimulation of the parathyroid hormone (PTH) receptor by PTH promotes translocation of β-arrestins 1 and 2 to the plasma membrane, association of the receptor with β-arrestins, internalization of receptor–β-arrestin complexes, and activation of extracellular signal–regulated kinase 1 (ERK1) and ERK2 (12). Accordingly, a selective agonist for β-arrestin signaling in the absence of G protein signaling promotes osteoblastic bone formation without stimulating bone resorption. This suggests the possibility that β-arrestins may be clinically effective as anabolic agents in the treatment of diseases that are characterized by insufficient rates of bone formation, such as osteoporosis (13).

GPCR Sensitivity and the Timing of Puberty

Distinct abnormalities of genes that encode GPCRs have been implicated in pubertal abnormalities (Table 2). Gain-of-function mutations in two distinct GPCRs have been associated with precocious puberty.

Table 2

GPCRs and their ligands implicated in pubertal disorders. GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone; hCG, human chorionic gonadotropin; FSH, follicle-stimulating hormone; nIHH, normosmic hypogonadotropic hypogonadism; CPP, central precocious puberty; FMPP, familial male-limited precocious puberty.

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In 1993, Shenker et al. (14) reported a germinative mutation of the luteinizing hormone (LH) receptor in a boy affected by sporadic or familial gonadotropin-independent precocious puberty, also known as testotoxicosis. Since then, several dominant, constitutively activating mutations in the LH receptor have been detected (8). All boys with this condition display elevated concentrations of testosterone. Whereas their concentrations of LH remain at a prepubertal level, they exhibit early signs of puberty, rapid virilization, and linear growth acceleration by 1 to 4 years of age. The LH receptor signaling pathway is prematurely activated in this condition because of its constitutive activation. In addition, a somatic gain-of-function mutation of the LH receptor is found in Leydig cell tumors, which leads to gonadotropin-independent precocious puberty (15, 16).

In 2008, Teles et al. (17) identified a nonconstitutive mutation of the kisspeptin receptor (KISS1R, also known as GPR54)—which is considered one of the gatekeepers of pubertal development in mammals—as a cause of central precocious puberty. Teles et al. found a unique heterozygous missense mutation (p.R386P) located in the region of the gene encoding the C-terminal tail of the KISS1R in a Brazilian girl with idiopathic central precocious puberty (CPP). In vitro studies revealed no substantial differences between the activities of the R386P mutant KISS1R and wild-type KISS1R in transfected cells under basal conditions, indicating that the R386P mutation does not generate a constitutively active receptor. Nevertheless, in repeated time-course studies, the rate of decline in inositol phosphate accumulation after stimulation of cells with kisspeptin, the ligand for KISS1R, was slower in cells transfected with plasmid encoding the R386P mutant KISS1R than in cells transfected with plasmid encoding the wild-type receptor, which resulted in the production of substantially higher amounts of inositol phosphates by the cells with the mutant receptor. Similarly, phosphorylation of ERK in cells with R386P KISS1R was prolonged relative to that in cells with wild-type KISS1R, confirming the extended activation of intracellular signaling pathways by the mutant receptor in response to kisspeptin. These findings demonstrate a reduction in the rate of desensitization of the mutant KISS1R. A previously uncharacterized missense mutation in the gene encoding the ligand of this receptor was identified in a boy with idiopathic CPP, who showed sexual development at 1 year of age (18). In vitro studies revealed that the p.P74S variant was associated with the resistance of kisspeptin to degradation relative to that of the wild-type protein, which suggests a role for this mutation in the precocious puberty phenotype.

Scarce documentation of the promiscuous activation of the follicle-stimulating hormone (FSH) receptor, a GPCR of the rhodopsin subfamily, suggests an uncommon mechanism of receptor activation. Heterozygous mutations in the transmembrane domain of FSH receptor have been identified in pregnant women with spontaneous and recurrent ovarian hyperstimulation (19, 20). These mutations result in hypersensitivity to human chorionic gonadotropin (hCG). The timing of onset and progression of symptoms coincides with the usual gestational fluctuations in the amounts of hCG, which suggests that hCG is a trigger for ovarian hyperstimulation syndrome. In vitro studies have confirmed promiscuous activation of FSH receptor by high concentrations of hCG; however, despite molecular investigation by several research groups, no other gain-of-function mutations have been identified in girls with gonadotropin-independent precocious puberty associated with autonomous ovarian cysts (21, 22).

Several loss-of-function mutations of distinct GPCRs cause hyper- or hypogonadotropic hypogonadism in humans (23). In this condition, lack of secondary sexual features, infertility, and osteoporosis are usually the chief complaints. An increasing number of GPCR mutations, including those in GnRH, kisspeptin, prokineticin 2, and neurokinin B receptors, are implicated in the pathogenesis of congenital isolated hypogonadotropic hypogonadism (2329). This highlights the importance of GPCRs in pubertal development and reflects the heterogeneity and complexity of the genetic basis of child maturation. Furthermore, loss-of-function mutations in the LH and FSH receptors result in testicular and ovarian resistance to gonadotropins, leading to different degrees of gonadal failure in both sexes (3034).

The highly variable timing of normal puberty suggests the involvement of multiple regulatory genes in this complex biological process. Early or delayed onset of puberty has psychosocial and public health implications. Indeed, early puberty is associated with increased long-term risk for diseases, including obesity, diabetes, and cancer (35). Whereas girls with earlier menarche (the first menstrual cycle) are heavier and taller than other girls during childhood, they remain heavier but shorter as adults because of the earlier cessation of growth. Studies of twins estimate that 44 to 95% of the variance in age at menarche may be heritable (36). Four independent genome-wide association studies of the timing of menarche show a common genetic variation in a gene on chromosome 6q21 (37). In addition, a pericentromeric region of chromosome 2 is associated with pubertal delay in families affected with constitutional delay of growth and puberty (38). In addition, polymorphisms in genes that encode GPCRs, such as KISS1R, are also associated with central precocious puberty (39).

GPCR Sensitivity in Bone and Mineral Metabolism

Circulating amounts of calcium ions (Ca2+) are maintained within a narrow physiological range, mainly by the action of the GPCR-stimulating PTH and the calcium-sensing receptor (CaSR), an inhibitory GPCR. During prenatal life, embryonic Ca2+ homeostasis is maintained by a GPCR that is stimulated by PTH-related protein (PTHrP). The latter has potent effects in growth plate cartilage and bone, through endocrine, paracrine, and autocrine mechanisms in prenatal through adult life, and in several diseases. Advances in the understanding of the mechanisms that regulate the sensitivities of receptors for PTH and PTHrP through gene expression, receptor desensitization, endocytosis, recycling, and intracellular signaling will help to elucidate the roles of these receptors in development, growth, and maturation (40).

Pseudohypoparathyroidism type Ia (PHP Ia) with Albright hereditary osteodystrophy is caused by a mutation that results in the loss of function of the Gαs isoform encoded by GNAS on the maternal allele and the resultant expression of only the paternal allele. In contrast, pseudopseudohypoparathyroidism (PPHP) is caused by mutations that result in loss of function of the Gαs isoform of the paternal allele of GNAS and the resultant expression of GNAS only from the maternal allele (41). As mentioned earlier, Gαs is ubiquitously expressed, mediating the cellular actions of numerous GPCR ligands; however, only a limited number of hormones that rely on Gαs signaling appear to be affected in PHP. This selectivity of hormone resistance suggests that Gαs mRNA from only one parental allele is transcribed in certain hormone-responsive cells. Gene polymorphism of GNAS may influence the sensitivity of receptors for PTH and PTHrP and the functions of effectors (42) and, as a consequence, the spectrum of variations in child growth and development.

Parathyroid cells can sense small fluctuations in the concentration of plasma Ca2+ by virtue of the CaSR. Several inherited human conditions are caused by inactivating or activating mutations of this receptor (43). The CaSR regulates processes such as cellular proliferation and differentiation, secretion, membrane polarization, and apoptosis in various cells and tissues (44). Gene polymorphism of the CaSR may influence receptor sensitivity to serum ionized calcium (45). Compounds that mimic and activate the CaSR and inhibit the secretion of PTH are known as calcimimetics. The newly discovered GPCR 48 (Gpr48) is an orphan receptor of unknown function. Gpr48 inhibits the production of osteocalcin, bone sialoprotein, and collagen (46). Deletion of Gpr48 in mice results in a delay in differentiation of osteoblasts and mineralization during embryonic bone formation (46). Postnatally, bone remodeling is also substantially affected in these mice (46).

GPCRs in Tissue Differentiation

The identification of mechanisms that underlie the spectrum and plasticity of developmental programming and maturation is a major goal in current child health research. GPCRs and their signaling targets function in diverse systems and stages of life, affecting homeostasis, neuronal sensory and signal transmission, and the differentiation of various cell lineages during development (47).

During embryogenesis, neuronal differentiation is coordinated by GPCR signaling pathways. The binding of adenosine triphosphate (ATP) to the purinergic GPCR P2Y2 activates the G protein Gq and PLC-β, which leads to the production of brain-derived neurotrophic factor (BDNF) and myocyte enhancer factor 2A, which in turn are important for neuronal development (48). Pituitary adenylyl cyclase–activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) act through their respective GPCRs to induce the differentiation of embryonic stem cells into the neuronal lineage (49, 50), and PACAP induces the differentiation of astrocytes. The chemokine stromal-derived factor 1 (SDF-1, also known as CXCL12) acts through its receptor CXCR4 to guide axonal growth (51). SDF-1 signaling through CXCR4 also controls the differentiation of glial cells, which provide support and nutrition to neurons in the developing and maturing nervous system (52).

The differentiation of several cell types is regulated by complex networks of signaling processes, with considerable involvement of GPCRs. Thus, smooth muscle cells are regulated by S1P and SPC; osteoblasts by PTH/PTHrP and PGF2Ar; and adipocytes by GPCR-41 and GPCR43, Eta, MC2R, and MC5R, as recently reviewed (47, 53). The regulation of such tissue differentiation and function is important for child growth, maturation, homeostasis, and body composition. Elucidation of these processes will contribute to our understanding of growth disorders and childhood obesity.

GPCRs as Potential Drug Targets

One of the special features of GPCRs is that they are highly druggable (3). Nonetheless, only a small percentage of the approximately 800 known GPCRs have been targeted for therapeutics. Extensive efforts are under way to discover new generations of drugs against GPCRs with specifically targeted therapeutic uses, including receptor activators, allosteric regulators, inverse agonists, and drugs targeting hetero-oligomeric complexes (54, 55). An example of new drugs are the pharmacochaperones, which are small hydrophobic molecules that can penetrate the cell membrane, bind to the nascent GPCR, and rescue or shuttle mutant and underexpressed GPCRs to the cell membrane (56). A cell-permeable, small-molecule agonist for a mutant human LH receptor was constructed. This specific LH receptor pharmacochaperone recruits the mutant LH receptor to the cell surface (57). Similarly, a cell-permeable, small-molecule GnRH antagonist can also be used to rescue poorly expressed GnRH mutant receptors (56). It is clear that these and similar proteins serve important functions in a number of physiological and pathophysiological processes, and they are emerging as potential drug targets.

Perspectives

GPCRs transduce signals from a diverse array of endogenous ligands and regulate almost every aspect of child growth, development, and maturation. Extensive and intensive efforts of the scientific community have led to the establishment of a number of GPCR signaling databases, including GPCRSDB at the Centre for Molecular and Biomolecular Informatics (CMBI) (formerly at the European Molecular Biology Laboratory), tinyGRAP Mutant Database at CMBI (formerly at Tromso, Norway), the Olfactory Database (ORDB) at Yale, Swiss-Prot, and the GPCRS Natural Variants Database (NaVa). Recent studies of the naturally occurring mutations and polymorphisms in the genes encoding GPCRs have contributed considerably to the current knowledge of the physiology and pathophysiology of developmental variations and disorders of growth and development. It is estimated that 40 to 50% of the currently used therapeutic drugs target GPCRs directly or indirectly (58); however, only a small percentage of the members of the vast GPCR superfamily have been considered as targets for drug development. The possibilities for new drugs relating to the broad spectrum of variation and disorders of child growth and maturation are immense. Considering the versatility of GPCRs in the regulation of physiological processes, it seems likely that abnormalities in the genes responsible for encoding these receptors will result in relevant alterations of distinct physiological processes.

Upcoming discoveries about the signaling mechanisms of GPCRs and previously uncharacterized ligands for orphan receptors will undoubtedly increase appreciation of the importance of GPCR signaling in child development, growth, and maturation. Increased realization of the therapeutic potential of GPCR-targeted medicines in the treatment of aberrations and disorders during development and maturation will inevitably follow.

References

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