The role of GPCRs in bone diseases and dysfunctions

The superfamily of G protein-coupled receptors (GPCRs) contains immense structural and functional diversity and mediates a myriad of biological processes upon activation by various extracellular signals. Critical roles of GPCRs have been established in bone development, remodeling, and disease. Multiple human GPCR mutations impair bone development or metabolism, resulting in osteopathologies. Here we summarize the disease phenotypes and dysfunctions caused by GPCR gene mutations in humans as well as by deletion in animals. To date, 92 receptors (5 glutamate family, 67 rhodopsin family, 5 adhesion, 4 frizzled/taste2 family, 5 secretin family, and 6 other 7TM receptors) have been associated with bone diseases and dysfunctions (36 in humans and 72 in animals). By analyzing data from these 92 GPCRs, we found that mutation or deletion of different individual GPCRs could induce similar bone diseases or dysfunctions, and the same individual GPCR mutation or deletion could induce different bone diseases or dysfunctions in different populations or animal models. Data from human diseases or dysfunctions identified 19 genes whose mutation was associated with human BMD: 9 genes each for human height and osteoporosis; 4 genes each for human osteoarthritis (OA) and fracture risk; and 2 genes each for adolescent idiopathic scoliosis (AIS), periodontitis, osteosarcoma growth, and tooth development. Reports from gene knockout animals found 40 GPCRs whose deficiency reduced bone mass, while deficiency of 22 GPCRs increased bone mass and BMD; deficiency of 8 GPCRs reduced body length, while 5 mice had reduced femur size upon GPCR deletion. Furthermore, deficiency in 6 GPCRs induced osteoporosis; 4 induced osteoarthritis; 3 delayed fracture healing; 3 reduced arthritis severity; and reduced bone strength, increased bone strength, and increased cortical thickness were each observed in 2 GPCR-deficiency models. The ever-expanding number of GPCR mutation-associated diseases warrants accelerated molecular analysis, population studies, and investigation of phenotype correlation with SNPs to elucidate GPCR function in human diseases.


INTRODUCTION
Bone development and bone remodeling are processes primarily governed by osteoblast, osteoclast, and chondrocyte differentiation and activity. Fetal bone development proceeds through two courses, intramembranous ossification (typical in flat bone formation) and endochondral ossification (primarily in long bones). Intramembranous ossification is largely influenced by mesenchymal cell differentiation into mature osteoblasts, 1 while endochondral ossification is driven by mesenchymal cell differentiation into chondrocytes, which then undergo hypertrophy. 2 Bone remodeling occurs throughout life and involves resorption of mature bone tissue by osteoclasts, which differentiate from hematopoietic cell precursors, 3,4 and new bone tissue formation by osteoblasts, which arise from mesenchymal stem cells (MSCs) 5,6 (Fig. 1). Each cell type is regulated by assorted hormones and paracrine factors. These factors determine the relative rates of bone formation and resorption, processes whose homeostasis is critical to prevent bone structure damage, and consequent metabolic bone diseases. 7 G protein-coupled receptors (GPCRs) are the most numerous transmembrane (TM) protein family implicated in multiple biological processes, including bone development and remodeling, 8,9 vision, 10 taste, 11 smell, 12 neurotransmitter signaling, 13 inflammation/immune response, 14 autonomic nervous system regulation, 15 homeostasis maintenance, 16 and tumor growth and metastasis. 17 Because GPCRs play important roles in physiological and pathological processes, have easily targeted ligand-binding domains, and bind diverse chemical modulators, they comprise the most important class of drug targets, accounting for 12% of all human protein drug targets and the therapeutic effects of approximately 34% of clinically used drugs. 18,19 Certain GPCRs and their signaling pathways are responsible for bone homeostasis, and disruption or mutation of these GPCRs results in human bone diseases or dysfunctions, [20][21][22][23][24][25][26][27][28][29] the majority of whose phenotypes have been validated in mouse models. 8,[30][31][32][33][34][35][36][37][38][39][40][41][42][43] Therefore, GPCRs are necessary for regulating bone development and remodeling.
More than 800 human GPCRs (approximately 2%-3% of all human genes) have been identified that share common structural motifs. Approximately 150 putative human GPCRs have still unknown functions with unknown ligands and are consequently GRK5, and GRK6). 57 GRK 1 and 7 expression is limited to the retina; GRK 2, 3, 5, and 6 are expressed ubiquitously; and GRK4 expression is predominantly observed in the brain, kidney, and testes. 58 GRKs terminate GPCR activation via phosphorylation of substrate intracellular loops and C-terminal tails. The phosphorylated GPCR then binds arrestins, which exclude G protein interaction and induce receptor-arrestin complex internalization, shutting down signal transduction. 59,60 Therefore, modulation of GRK protein stability is a potential feedback mechanism for regulating GPCR signaling and basic cellular processes.
Arrestin family proteins regulate GPCR signal transduction 61,62 by terminating G protein signaling and initiating arrestinmediated GPCR downstream cascades. Mammalian cells express four arrestins: arrestin-1 (also known as visual arrestin), arrestin-2 (also known as β-arrestin 1), arrestin-3 (also known as β-arrestin-2), and arrestin-4 (also known as cone arrestin). Arrestin-1 and arrestin-4 are selectively expressed in the retina, and arrestin-2 and arrestin-3 have a broad expression pattern in various cell types. Arrestin-2 and arrestin-3 are~80% identical in sequence and have overlapping roles in GPCR regulation. [63][64][65][66] As GPCRs have a variety of signaling modalities that can selectively stimulate (or inhibit) intracellular signaling pathways to treat different diseases by biased signaling, which can minimize the risk of side effects, 67,68 GPCRs have been major targets of modern therapeutics. For example, the rhodopsin family GPCR Angiotensin II (AngII) type I receptor (AT1R) has been targeted for the treatment of cardiovascular diseases. 69,70 Recently, AT1R was shown to activate both Gαq signaling and β-arrestin signaling to exert different functions and side effects. Therefore, the β-arrestinbiased ligand TRV027 for AT1R is currently in a phase II clinical trial. TRV027 specifically activates AT1R-β-arrestin signaling (associated with increased cardiomyocyte contractility and cardiac apoptosis prevention) but without stimulating Gαq signaling, which is linked to vasoconstriction and sodium and fluid retention. 71,72 Multiple GPCRs exhibit bone expression, 73 and GPCR signaling regulates the proliferation, differentiation, and apoptosis of osteoblasts, osteoclasts, and chondrocytes. 6,[73][74][75][76] GPCRs signal through several canonical pathways to regulate osteoblast function 77 : the Gs and Gi pathways regulate AC, increasing or decreasing intracellular cAMP levels, respectively, while Gαq Fig. 1 Bone cells and bone remodeling. Bone is continuously remodeled to maintain tissue integrity. Remodeling begins with old bone resorption by osteoclasts, which differentiate from hematopoietic stem cells. Following resorption, unclassified macrophagelike cells, which are also from hematopoietic stem cells, are found at the remodeling site in the intermediate or reversal phase. Osteoblast precursors, which arise from mesenchymal stem cells, are then recruited and proliferate and differentiate into mature osteoblasts and secrete new bone matrix. The matrix then mineralizes to generate new bone, completing the remodeling process activates phospholipase C (PLC) to increase intracellular calcium. 73,[78][79][80][81][82] In addition, GRK phosphorylation and β-arrestin signaling govern osteoblast function [83][84][85] (Fig. 3). Recent advances have shed light on the mechanisms of osteoclast 9,76,86,87 and chondrocyte [88][89][90][91][92] differentiation and function; however, how GPCR signaling regulates osteoclasts and chondrocytes remains largely unknown. The expression of multiple GPCRs by different bone cells and the activation of multiple signaling pathways by a single GPCR, together with the wide variety of GPCRs and the signaling redundancy often seen downstream of GPCR activation, pose significant challenges to clarifying a given GPCR's function in bone development and disease. Nevertheless, incremental advances into the in vivo roles of GPCR signaling pathways and their effects on bone biology have been recently attained (Fig. 2).

DISEASES OR DYSFUNCTION CAUSED BY GPCR MUTATION OR DELETION IN HUMANS AND MICE
Glutamate family Glutamate receptors are predominantly expressed by neuronal and glial cells 93 and transmit glutamate-mediated postsynaptic excitation of neural cells. They regulate neural communication, memory formation, and learning. Several diseases in humans have an established association with glutamate receptor gene mutations, including Parkinson's disease, 94 Huntington's disease, 95 ischemic stroke seizures, 96 attention deficit hyperactivity disorder, 97 addiction, 98 and autism. 99 There are two types of glutamate receptors: metabotropic receptors (mGluRs) bearing a single 7TMD and multimeric ligandgated ion channels, and ionotropic receptors (iGluRs). 100 The mGluRs are linked to G protein complexes whose associated GTPase activity mediates their signaling. Upon binding glutamate, mGluRs initiate G protein activation as described above, triggering intracellular signaling cascades. 101 The iGluRs are a composite family, including the kainate (Ka), N-methyl-D-aspartate (NMDA), and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) groups. 102 The different iGluRs have different properties and kinetics, with AMPA and kainates predominantly active in Na + and K + permeability, while NMDA is predominantly active Ca 2+ in permeability. 100 A variety of glutamate receptors have abundant bone expression and function in bone remodeling. [103][104][105][106][107] One such receptor is an essential regulator of calcium homeostasis, the calcium-sensing receptor (CASR). Under physiological Ca +2 levels, CASR is activated by extracellular calcium and inhibits parathyroid hormone (PTH) and PTH-related protein (PTHrP) secretion. If systemic calcium levels drop, CASR signaling decreases, allowing PTH and PTHrP secretion, which induces renal retention of Ca +2 , increased gut Ca +2 absorption, and eventually elevated bone resorption. 108,109 Lorentzon et al. found that different CASR alleles are related to bone mineral density (BMD), 110 and healthy adolescent girls with the S allele have lower BMD than individuals lacking the S allele, and Di et al. 20 also verified that the CASR A986S polymorphism increased the risk of osteoporosis in aging males. Knockout of Casr in osteoblasts, driven by 2.3Col(I)-Cre or OSX-Cre, resulted in reducing BMD and bone length to block mouse skeletal development. 88 Moreover, knockout of Casr, driven by Col(II)-Cre, in chondrocytes blocks embryonic development and cartilage maturation. 88 Additionally, the mice with global knockout of Casr showed a significantly reduced body length. 30 Additional phenotypes were validated in mouse models, in which deletion of Gababr1, 111 Gprc6a, 112,113 and Grm1 114 reduced mouse BMD, while Tas1r3 deficiency impaired osteoclast function, resulting in reduced bone resorption and increased bone mass. 115,116 Gababr1-null mice reduce BMD primarily through negatively regulating BMP and upregulating RANKL to affect bone remolding, 111 while the effects of Gprc6a deletion were primarily caused by defective osteoblast-mediated bone mineralization. 112,113 Grm1 knockout mice exhibit enhanced bone maturation, marked by premature growth plate fusion, shortened long bones, and lower BMD 114 (Table 1).

Rhodopsin family
The rhodopsin family (class A in the A-F classification system), which includes 701 members in humans, is the largest vertebrate GPCR family and regulates many processes throughout the body. Rhodopsin receptors are structurally different from other GPCR subfamilies as they generally possess short N-termini. 47 The ligands for most rhodopsin receptors, though diverse in structure, typically bind a cavity between the TM regions, 117 whereas in other GPCR families, the N-terminus plays a key role in ligand binding. Important exceptions exist, particularly the glycoproteinbinding receptors (lutropin, follitropin, and thyrotropin), which bind ligands through an N-terminal domain.
Based on experimental phylogenetic investigation, there are four main groups of rhodopsin GPCRs (α, β, γ, and δ), which are subdivided into 13 subgroups in humans. 46 The α-group includes five branches: the prostaglandin, amine, opsin, melatonin, and MECA receptor clusters. The β-group includes 36 receptors without any main branches. The γ-group contains three main branches: the SOG, MCH, and chemokine receptor clusters, while the four branches of the δ-group are the MAS-related, glycoprotein, purin, and olfactory receptor clusters. 46 The rhodopsin family α-group. When the α-group rhodopsin GPCRs were analyzed for effects of mutation or deletion, eight GPCRs were associated with human bone diseases or dysfunctions. Mutations of ADRB2, 118 CNR2, 21,119,120 and DRD4 121,122 were associated with reduced human BMD, while MC4R 123 increased BMD. ADRB2 genotypes AG and GG had more frequent osteoporosis at the femoral neck (3.27 and 3.89 times more frequent, respectively, compared to AA genotype) in a study of 592 postmenopausal Korean women. 118 Woo et al. suggested that the CNR2 gene polymorphisms rs2501431, rs3003336, rs2229579, and rs4237 may affect BMD in postmenopausal Korean women. 119 A CNR2 polymorphism is associated with low BMD in Japanese 120 and French women. 21 Japanese men with the 521C>T polymorphism of DRD4 more frequently had reduced BMD, but no difference   (Table 2). DRD2 polymorphism could influence human height in childhood, acting through the hypothalamus (growth hormone (GH)releasing hormone)-pituitary (GH)-Insulin-like growth factor 1 (IFG-1) axis, 22 while MTNR1B polymorphism was associated with adolescent idiopathic scoliosis (AIS). Moroca et al. found that, compared with CC (MTNR1B) (rs4753426), the risk of AIS significantly increased in Hungarians bearing the CT allele. 24 Gary et al. reported lower fracture incidence among elderly Swedish women bearing the MC4R C-allele. 124 Curiously, lipocalin 2, a recently identified ligand of MC4R, is secreted by osteoblasts in mice and signals to suppress appetite by binding MC4Rexpressing hypothalamic neurons 125 ; MC4R polymorphisms have also been associated with early-onset obesity. 126 Mutation of CNR2 21 and MTNR1B 127 had an additional association with human osteoporosis. Karsak et al. found that two missense variants (the double single-nucleotide polymorphism (SNP) rs2502992-rs2501432 and Gln63Arg; rs2229579 and His316Tyr) are associated with osteoporosis in postmenopausal Caucasian women, 21 while Li et al. found that MTNR1B rs3781638 is associated with osteoporosis in Chinese geriatrics. 127 The ADRB2 polymorphism (rs1042714) was also associated with heterotopic ossification in adult trauma patients with fractures. 128 EDG2 26 and H4R 23 were associated with human osteoarthritis (OA) in Japanese people. EDG2 SNPs (rs3739708) affect AP-1 transcriptional activity, which may increase EDG2 expression when the allele is upregulated in knee OA patients, while Yamaura et al. found higher expression of H4R mRNA in synovial tissues from patients with OA (Table 2).
Eighteen α-group GPCR genes have been reported to cause bone dysfunctions when deleted in mouse models. The deletion of A1r, [129][130][131] 133 and Mc4r 137 knockout mouse bone mass and BMD were significantly increased, accompanied by impaired bone resorption; Mc4r-deficient mice also had higher CART expression, and deleting one CART allele ameliorated the bone resorption phenotype, suggesting that Mc4r function in hypothalamic neurons may regulate osteoclast function, 149 although direct synovial and bone functions for proopiomelanocortin-derived peptides have been reported. 150 Deletion of EP1 135 increased bone mass and BMD by promoting osteoblast-mediated bone formation. A2ar, 139,140 A2br, 141,142 Adrb1, 118,143,144 Adrb2, 143,144 Lpar1, 32,148 and Ep1 135 knockout in mice induced bone loss by promoting bone resorption and suppressing bone reformation, while Htr2 deletion suppressed osteoblast recruitment and proliferation and led to osteopenia. 147 Htr2 147 and Ep1 135 also participate in regulating nervous systemmediated bone loss.
The deletion of Cnr2 increased mouse body length by regulating growth plate chondrocyte function, 151 while Lpar1 reduced body length by regulating osteoblast function. 32 Furthermore, M3R deletion caused mouse osteoporosis by altering osteoblast and osteoclast function or neuronal regulation, 33,34,122 H4r deletion accelerated mouse rheumatoid arthritis by promoting osteoclastogenesis, 152 and Mc1r deficiency caused an articular cartilage phenotype accompanied by accelerated surgically induced murine OA. 136 Deletion of A3ar promoted mouse osteosarcoma cell proliferation, tumor formation, and metastasis, mainly by activating the protein kinase A (PKA)-Akt-nuclear factor (NF)-κB axis. 25 Ep1 deletion accelerated fracture repair by enhancing osteoblast differentiation, 153 and Ep2 deletion reduced mouse bone stiffness, which may be caused by stimulating cAMP formation, an early cellular signal that stimulates bone formation. 154 Ep4 deletion inhibited mouse bone resorption, though the reason is disputed, with one paper claiming it was a cAMPdependent mechanism 155 or through proinflammatory cytokines and lipopolysaccharides. 155,156 Cnr2 deletion reduced mouse agerelated or ovariectomy-induced bone loss by osteoclast inhibition. 157,158 Moreover, while Cnr2 knockout reduced bone mass in C57BL/6 mice by regulating osteoblastogenesis and osteoclastogenesis, 31,159 the opposite phenotype was found in CD1 mice, which had increased bone mass. 160 These results suggest that different GPCRs have different physiological functions to regulate bone remodeling, and even the same gene may have different physiological functions regulating bone remodeling in different strains of mice ( Table 2).
The β-group of the rhodopsin family. Analysis of the effects of rhodopsin β-group GPCR mutation or deletion uncovered 10 GPCRs associated with bone diseases or dysfunctions. Of particular interest is the ghrelin receptor, GHSR, whose mutation was associated with reduced human height. 27 Normally, ghrelin secreted by the stomach induces appetite and regulates lipid metabolism. In 2 families with familial short stature, Pantel and coworkers identified a GHSR missense mutation that downregulated receptor protein levels and selectively impaired GHSR constitutive activity without affecting its response to ghrelin. In Ghsr-deficient mice, a reduction in BMD was caused by impaired bone formation, although the mechanism is disputed. In one report, the phenotype was due to acylated ghrelin signaling and was partially suppressed by unacylated ghrelin 161 ; more recently, Gshr re-expression in the osteoblasts, but not in the osteoclasts, of Gshr −/− mice was able to restore bone formation by promoting osteoblast differentiation. 162 Additional β-group rhodopsin GPCRs implicated in human bone disorders, including GNRHRs, 28 were

TAS1R3
Mouse Reduced bone resorption and increased bone mass Eaton et al. 115 Simon et al. 116 BMD bone mineral density, GPCR G protein-coupled receptor The role of GPCRs in bone diseases and dysfunctions J Luo et al.  129 Kara et al. 130 Kara et al. 131

A2AR
Mouse Reduced bone mass and inhibited bone formation Mediero et al. 139 Mediero et al. 140

A2BR
Mouse Reduced BMD and bone mass Corciulo et al. 141 Carroll et al. 142

ADRB1
Mouse Reduced bone mass and BMD Pierroz et al. 143 Bonnet et al. 144

ADRB2
Human SNP associated with reduced BMD, increased risk of fractures, and heterotopic ossification Lee et al. 118 Mitchell et al. 128 Mouse Reduced bone mass and BMD Pierroz et al. 143 Bonnet et al. 144

CNR1
Mouse Increased trabecular bone mass Tam et al. 132 Idris et al et al. 133 Khalid et al. 134

CNR2
Human The rs2501431, rs3003336, rs2229579, and rs4237 polymorphisms associated with osteoporosis and decreased BMD Woo et al. 119 Yamada et al. 120 Karsak et al. 21 Mouse Reduced bone mass in C57BL/6 background Ofek et al. 31 Sophocleous et al. 159  Mouse Reduced bone mass and bone formation Kumar et al. 145 Yadav et al. 146 Collet et al. 147

LPAR1
Mouse Reduced body length and bone mass Gennero et al. 32 David et al. 148

M3R
Mouse Induced osteoporosis and reduced BMD Shi et al. 122 Lips et al. 33 Kauschke et al. 34 24 Li et al. 127 AIS adolescent idiopathic scoliosis, BMD bone mineral density, GPCR G protein-coupled receptor, SNP single-nucleotide polymorphism The role of GPCRs in bone diseases and dysfunctions J Luo et al.
associated with reduced human BMD and short stature, and EDNRA was associated with abnormal human tooth development. 163 Homozygous partial loss-of-function mutations in GNRHRs caused the reduction in height and BMD through delayed puberty or isolated hypogonadotropic hypogonadism. 28 The EDNRA (rs1429138) gene polymorphism affected gene expression during early craniofacial development and was associated with abnormal human tooth development. 163 Additional phenotypes were identified in GPCR knockout mouse models. The deficiency of Avpr1a, 164 Npy1r, 165,166 and Npy2r 167-173 increased mouse bone mass and BMD, while Cckbr, 174,175 Ghsr, 161 and Npy6r 176 deficiency reduced bone mass and BMD. Tama et al. reported a dramatic bone mass increase in Avpr1α −/− mice resulting from elevated bone formation and reduced resorption, 164 while Npy1r 165,166 and Npy2r 167-173 mice directly regulate osteoblast activity and bone formation; BMD changes occur when these genes are deleted. 165 In contrast, mice deficient in Cckbr had reduced bone mass and BMD by disrupted calcium homeostasis. 174,175 Npy6r deletion in mice suppressed osteoblast numbers, osteoid surface area, and bone mineralization while stimulating osteoclast formation and bone resorption, presumably via a suprachiasmatic nucleus relay due to the narrow range of cells that expresses this receptor. 176 Furthermore, Oxtr deletion caused mouse osteoporosis by inhibiting the differentiation of osteoblasts and stimulating osteoclast formation, 35 and Ednra deletion caused mouse mandibular and craniofacial defects, possibly by regulating Dlx5 and Dlx66, which are downstream mediators of Ednra signaling. [177][178][179][180][181] Fracture repair was delayed while bone callus volume and callus strength decreased in osteoblast-specific Npy1r knockout mice, 182 and Gpr120 deletion promoted osteoblastic bone formation and negatively regulated osteoclast differentiation, survival, and function 183,184 (Table 3).
The rhodopsin family γ-group. Among the γ-group rhodopsin GPCRs, two GPCR gene polymorphisms were associated with human bone diseases or dysfunctions (Table 4). Eraltan et al. found CCR2 V64I gene polymorphisms in postmenopausal women and demonstrated a positive association of CCR2 Val/Ile and CCR2 Val+ genotypes with osteoporosis risk. 185 This polymorphism appears to increase CCR2 protein half-life 186 and may also be associated with cancer risk and other diseases. [186][187][188] Furthermore, Lu and coworkers discovered that three OPRM1 SNPs (rs9479769, rs4870268, and rs1998221) were nominally associated with hip, spine, and whole-body BMD phenotypes in female American Caucasians, potentially via effects on alcohol consumption and/or estrogen signaling. 29 Fourteen genes from the γ-group GPCRs have been reported to cause bone dysfunctions in knockout mouse models. The deficiency of Cx3cr1 189 increased mouse bone mass and BMD by regulating both osteoblasts and osteoclasts, while deficiency of Bdkrb1, 190 Ccr1, 191,192 Ccr6,193 Cmklr1,194 Cxcr2, 36 Cxcr4, 195 Gpr1, 196 and Gpr54 197 reduced bone mass and BMD. Deletion of Bdkrb1 increased mouse bone loss and the number of osteoclasts by increasing differentiation into functional osteoclasts, 190 and deficiency of Ccr1 191,192 and Gpr1 196 caused osteopenia due to decreased osteoclast and osteoblast activity. Doucet et al. 193 found that Ccr6 −/− mice exhibited significantly decreased trabecular bone mass and reduced osteoblast numbers. Mouse Induced mandibular and craniofacial defects Ruest et al. 177 Ruest et al. 178 Sato et al. 179 Tavares et al. 180 Clouthier  167 Baldock et al. 168 Shi et al. 169 Allison et al. 170 Sainsbury et al. 171 Sainsbury et al. 172 Lundberg BMD bone mineral density, GPCR G protein-coupled receptor, SNP single-nucleotide polymorphism The role of GPCRs in bone diseases and dysfunctions J Luo et al.
Mechanistic studies indicated that Ccr6 loss delayed osteoblast marker gene expression, inhibited osteoblast differentiation, and reduced mineralization. Zhao et al. 194 found that Cmklr1 deficiency disrupted the balance between osteoblastogenesis and osteoclastogenesis, causing MSCs to shift from osteogenic to adipogenic differentiation and enhancing osteoclast formation and consequently lower bone mass in male mice. Zhu et al. 195 found that osteoprecursor-specific inactivation of Cxcr4 impaired osteoblast development and reduced postnatal bone formation, leading to a reduction in BMD and femoral length. Conversely, a decrease in BMD and body length in Cxcr2 −/− mice occurred despite no alteration in bone formation or bone resorption. 36 Furthermore, the Mchr1 −/− mice have osteoporosis caused by elevated bone resorption resulting in a reduction in the cortical bone mass, while trabecular bone was unaffected. 198 Ccr2 deficiency reduced macrophage infiltration and impaired osteoclast function, thus delaying bone fracture healing, 199 while Cxcr4 knockout mice delayed bone fracture healing by inhibiting osteoblastogenesis. 200 Cxcr2 knockout mice had attenuated autoantibody-mediated arthritis caused by a function of Cxcr2 neutrophil recruitment, 201 while Gpr142 knockout mice showed reduced arthritis scores and disease incidence in an anti-type II collagen antibody-induced arthritis model alongside decreased inflammatory cytokine production. 202 Mader et al. found that while Ccr2 −/− mice had larger and stronger bones than wild-type mice, they reported that Ccr2 loss did not significantly protect against bone loss due to disuse or estrogen loss. 203 Ccr5 deletion was linked to reduced cartilage degeneration postsurgery without significant changes in the degree of synovitis and bone metabolic parameters 204 and promoted osteoclast function in orthodontic tooth movement. 205 Furthermore, Ccr7 deletion reduced functional deficits and subchondral bone changes in a surgical destabilization of the medial meniscus model, suggesting that certain chemokine receptors may directly affect nociception 206 (Table 4).
The δ-group of the rhodopsin family. Five human bone diseases or dysfunctions were associated with eight δ-group rhodopsin GPCR gene polymorphisms. Mutation of LHCGR 207-209 was associated with reduced human height; FSHR, 210 RXFP2, 211 and TSHR 212 mutations were associated with human osteoporosis; OR2H1 was associated with human OA 213 ; FSHR, 210 LGR4, 214 RXFP2, 215 and TSHR 216 were associated with reduced human BMD, and FPR mutation was associated with juvenile periodontitis (Table 5). Shenker et al. 209 found eight different families with the same A>G base change that substitutes glycine for aspartate at LHCGR amino acid 578. This mutation elevated cAMP levels when transfected into COS-7 cells, suggesting constitutive luteinizing hormone receptor activation, and was correlated with precocious puberty and increased male height. Rendina et al. 210 found that women with AA rs6166 (FSHR) had a higher postmenopausal osteoporosis risk than those carrying the GG rs6166 variant, and Ferlin et al. 210 found that young men with a T222P mutation in RXFP2 were at high risk of osteoporosis, while Liu et al. 212 suggested that an SNP (C-to-G substitution at codon 727) in TSHR may be an osteoporosis risk factor. Two SNPs in OR2H1 (rs1233490 and rs2746149) were suggestively associated with rheumatoid arthritis phenotypes. 213 Furthermore, the SNP rs6166 of FSHR significantly influenced postmenopausal female BMD, 210 the T222P mutation of RXFP2 was associated with a high risk of reduced young adult BMD, 215 and the TSHR-Asp727Glu polymorphism was associated with femoral neck BMD in elderly Caucasians. 216 Finally, two FPR mutations were found in juvenile periodontitis patients: one thymine-to-cytosine substitution at base 329 and the other a cytosine-to-guanine substitution at base 378. 217 Increasing evidence supports the FSHR subfamily member LGR4 in bone development. In humans, a rare nonsense mutation within LGR4 (c.376C>T) is strongly correlated with diminished BMD, 214 in accord with similar phenotypes in Lgr4 −/− mice. 8,9  BMD bone mineral density, CAIA collagen antibody-induced arthritis, DMM destabilization of the medial meniscus, GPCR G protein-coupled receptor, SNP single-nucleotide polymorphism The role of GPCRs in bone diseases and dysfunctions J Luo et al.
Furthermore, Lgr4 negatively regulates osteoclast differentiation by binding RANKL and downregulating RANK expression in mouse and human cells. 9 In vitro studies support Lgr4 regulation of osteoblasts and bone MSCs. 8,218 Mice treated with the Lgr4 extracellular domain to inhibit Lgr4 signaling had lower osteoporosis induced by RANKL injection or ovariectomy, 9,219 suggesting this GPCR as a potentially valuable therapeutic target in several bone diseases. Deletion of 16 δ-group GPCR genes caused bone dysfunctions in mouse models: deficiency of Ebi2, 86 Gpr55, 220 Gpr68, 221 P2y6, 222 and Ptafr 42 increased mouse bone mass and BMD; while Gpr65, 223 Gpr103, 224 Lgr4, 8,9 P2y1, 225 Rxfp2, 211,215 Tshr 37 reduced bone mass and BMD; and P2y12 -/mice had reduced age-associated bone loss with lower osteoblast activity, 226 while deletion of Par2 227 bone prevented periodontal disease in mice. Defective Ebi2 signaling suppressed osteoclast precursor cell migration to bones, which led to increased male mouse bone mass and protection of female mice from osteoporosis due to age or estrogen deficiency. 86 Gpr55 −/− mice had a significant increase in BMD due to stimulated osteoclast function, 220 and BMD was increased in Gpr68 −/− mice by increasing bone turnover and a shift toward increased bone formation over resorption. 221 The long bones and spine in P2y6r −/− mice exhibited increased bone mineralization, cortical bone volume, and cortical thickness caused by suppressing osteoclastogenesis, whereas trabecular bone parameters were unaffected. 222 Hikiji et al. 42 found that Pafr knockout suppressed bone resorption, thus preventing bone loss in ovariectomized (OVX) mice. In contrast, Gpr65 −/− mice had elevated OVX-induced bone loss induced with enhanced osteoclast formation and osteoclastic calcium resorption. 223 Gpr103 −/− mice had lower trabecular bone density, possibly from suppressing osteoblast-mediated bone formation, and the kyphosis phenotype was also found in Gpr103 knockout female mice. 224 P2y1 deletion reduced mouse BMD in part through increasing osteoclast formation and activity via ATP and ADP. 225,228 Rxfpdeficient mice presented with lower bone mass and a reduction in bone turnover via disrupted regulation of osteoblastogenesis and osteoclastogenesis. 211,215 The BMD reduction in Tshr −/− mice was caused by altering the regulation of both bone formation and resorption. 37 Keratinocyte-specific deletion of Par2 prevented Mouse Induced kyphosis and reduced BMD and bone mass Baribault et al. 224 LGR4 Human Mutation of c.376C>T was associated with reduced BMD Styrkarsdottir 214 Mouse Reduced body length and bone mass Luo et al. 8 Luo et al. 9 LHCGR Human A single A>G base change at position 578 was associated with reduced male height Soriano et al. 207 Bertelloni et al. 208 Shenker

RXFP2
Human T222P mutation was associated with osteoporosis and reduce BMD Ferlin et al. 211 Ferlin et al. 215 Mouse Reduced bone mass Ferlin et al. 211 Ferlin et al. 215 TSHR Human A C-to-G substitution at codon 727 was associated with osteoporosis and reduced BMD Liu et al. 212 Van et al. 216 Mouse Induced osteoporosis and reduced femur length and BMD Abe et al. 37 BMD bone mineral density, GPCR G protein-coupled receptor, SNP single-nucleotide polymorphism The role of GPCRs in bone diseases and dysfunctions J Luo et al.
periodontal bone loss by suppressing the inflammatory cascade, ultimately inhibiting osteoclast differentiation and activity. 227 Tshr knockout mice only reduced femur length, 37 while P2y13 −/− mice had increased tibia and tail length, 229 and Par2 deletion alleviated mouse arthritis. 230 Furthermore, several GPCR gene knockout mice displayed different phenotypes in different strains. The bone mass was reduced in young (4-week-old) P2y13-knockout mice via promotion of osteoblastogenesis and suppression of osteoclastogenesis, but mature (>10-week-old) P2y13-knockout mice showed the opposite bone phenotype via suppression of osteoblastogenesis. 229,231 P2y2 deficiency increased mouse bone mass in C57BL/6 mice 225,232 by promoting bone reformation and suppressing bone resorption but exhibited reduced bone mass in SV129 mice 233 by reducing osteoblast differentiation and mineralization. P2y7 knockout reduced bone mass in mixed genetic mice (129/ OlaXC57BL/6XDBA/2) by reducing osteoblast number and activity 234 but increased cortical thickness in C57 mice 235 promoting osteoclast-mediated bone resorption (Table 5).

Adhesion family
The adhesion GPCR family, including 33 human and 31 mouse GPCRs 236 (also referred to as family B 45 , B2, 237 EGF-TM7 receptors, 238 or the LNB-TM7 family 239 ), is the second largest subgroup of GPCRs. The adhesion GPCRs are divided into nine distinct subfamilies that share typical adhesion GPCR features. 240 The nine subfamilies are ADGRL (latrophilins), ADGRA, ADGRC (CELSRs), ADGRD, ADGRG, ADGRV (GPR98), ADGRE (EGF-TM7), ADGRF, and ADGRB (BAIs). 236 Adhesion GPCRs typically have an extensive N-terminal extracellular region featuring various domains that interact with the extracellular environment to execute adhesive functions. 241 Each receptor subfamily has a specific combination of domains in its N-terminal extracellular region. Receptors within a subfamily have differing numbers of domain repeats, with consequent variation in their N-terminal extracellular region. 241 A feature unique to adhesion family GPCRs is their autoproteolytic cleavage at the GPCR proteolysis site, 242,243 which occurs in the conserved GPCR autoproteolysis-inducing (GAIN) domain. 244,245 Autoproteolysis splits the highly glycosylated Nterminal fragment (NTF) from the membrane-spanning C-terminal fragment (CTF), which contains the canonical 7TM domain and the intracellular domain. The extracellular NTFs function similar to adhesion proteins, while CTFs activate intracellular signaling cascades. 240 Adhesion GPCRs are essential components in developmental processes. 246 Human adhesion GPCR mutations take part in nervous, bone, and cardiovascular disorders and cancers of all major tissues. [247][248][249] Analysis of human adhesion GPCR SNPs revealed four GPCRs that were associated with human bone diseases or dysfunctions. However, only two adhesion GPCR knockout animal models with bone phenotypes have been reported. The mutation of GPR126 was associated with alterations in AIS, 248,250-253 human height, [253][254][255][256][257] arthrogryposis multiplex congenital, 258 and aggressive periodontitis. 259 Xu et al. 252 found that three intronic SNPs of GPR126 (rs6570507, rs7774095, and rs7755109) were significantly associated with AIS in Chinese populations, and Kou et al. 253 also found that rs6570507 was the most significantly linked SNP to AIS in Japanese and European ancestry populations. Liu et al. found that SNPs rs6570507, rs3748069, and rs4896582 were associated with human height in Australian twin families, 256 and rs6570507 was also correlated with trunk length in a European GWAS metaanalysis. 257 Ravenscroft et al. 258 found that a missense substitution (p. Val769Glu [c.2306T>A]) impaired GPR126 autoproteolytic cleavage, resulting in reduced peripheral nerve myelination, possibly causing severe arthrogryposis multiplex congenital, and Kitagaki et al.'s study 259 in the Japanese population found that the GPR126 SNP rs536714306 impairs signaling and BMP2, ID2, and ID4 expression, negatively influences periodontal tissue, and leads to aggressive periodontitis, suggesting that bearers have an elevated risk for aggressive periodontitis. High GPR56 expression is correlated with positive rheumatoid factor levels in rheumatoid arthritis patients 260 and with the proliferation and invasion capacity of osteosarcoma cells. 261 Liu et al. found that knockdown of GPR110 can decrease human osteosarcoma cell proliferation, migration, and invasion capacity, suggesting a role of GPR110 in tumor progression and possible value as a novel prognostic biomarker in osteosarcoma. 262 Finally, Tonjes et al. found that two GPR133 variants (rs1569019 and rs1976930) were linked to adult height in Sorbian individuals, 263 in accord with a study that reported a microdeletion at 12q24.33, approximately 171.6 kb downstream of GPR133, which influences height in the Korean population. 264 In animal models, cartilage tissue-specific Gpr126 deletion caused idiopathic scoliosis and pectus excavatum accompanied by annulus fibrosis development in the intervertebral discs and increased chondrocyte apoptosis. Gpr126 was postulated to signal via upregulation of Gal3st4 transcription without altering intracellular cAMP. 253,265 Furthermore, Cd97 deficiency increased mouse bone mass, decreased osteoclast number, 266 and reduced arthritis 267 (Table 6).
Frizzled/Taste2 family The Frizzled/Taste2 receptors span two distinct clusters: the frizzled receptors (11 in both humans and mice) and the TAS2 receptors (25 human and 34 mouse). 46,268 Although obvious receptor similarities between these different branches are lacking, several features that differ from the other four GPCR families are shared among the sequences from this family of GPCRs, for example, IFL in TM2, SFLL in TM5, and SxKTL in TM7. The Frizzled receptors are highly conserved evolutionarily, while Taste2 GPCRs probably rapidly evolved and expanded in number. 47 The ten Frizzled receptors, FZD1-10, plus SMOH, are conserved in most mammals, with highly similar primary amino acid sequences, making the Frizzled family the most highly conserved GPCR family. 269,270 Frizzled GPCRs are Wnt receptors that play key roles in organism development, diseases and cell signaling. [271][272][273][274][275][276][277] Frizzled GPCRs have a CRD/FZ or FZ domain with ten conserved cysteines. The TAS2 receptors are not related to the glutamate receptor family's TAS1 receptors. TAS2 receptors have seven hydrophobic regions considered putative TM domains, but their very short N-terminal regions are unlikely to bind ligands. 278 All 25 functional human TAS2 genes (hT2Rs) are expressed in taste receptor cells of the human gustatory papilla. 279 DNA polymorphisms in 25 functional hT2R genes are relatively common, featuring a large number of amino acid substitutions. 280,281 Analysis of the human Frizzled/Taste2 family GPCR SNP revealed three GPCRs that were associated with human bone diseases or dysfunctions, and only three GPCR knockout animal models with bone phenotypes have been reported to date. Two FZD1 promoter SNPs (rs2232157, rs2232158) were linked to femoral neck area BMD in men of African ancestry. 282 [284][285][286] Mutation of frizzled-9 was associated with reduced human BMD. 273,287 Furthermore, Frojmark et al. reported that approximately 50% of male Fzd6 −/− mice displayed abnormal claw morphology or lack of claws, potentially by suppressing either WNT-3A-FZD or WNT-5A-FZD signaling. 284 Curiously, this phenotype was absent in female mice. Frizzled-9 knockout induced mouse osteopenia by reducing osteoblast-mediated bone formation 288 and reduced new bone formation after fractures by disturbing osteoblast function. 289 Smoh knockout reduced BMD, body length, and bone callus formation by reducing osteogenic differentiation in mice 38,290 (Table 7).
The role of GPCRs in bone diseases and dysfunctions J Luo et al.

Secretin family
The secretin receptor family has 15 members divided among four subgroups: CRHRs/CALCRLs, PTHRs, GLPRs/GCGR/GIPR, and GHRHR/PACAP/SCTR/VIPR. 46 These GPCRs are characterized by six conserved N-terminal domain cysteines and by seven conserved TM helices. [291][292][293] The N-terminal extracellular domain recognizes the secretin C-terminus, 291,294,295 with the conserved cysteines required for receptor function. 296 The secretin family GPCRs bind paracrine or endocrine peptide hormones (typically 30-40 amino acids long 297 ), often indiscriminately. Secretin GPCRs regulate diverse physiological responses, including the cell cycle, differentiation, proliferation, and additional endocrine hormone release. Secretin GPCRs generally signal through AC and to a lesser extent through PLC and intracellular calcium mobilization, although they are not confined to these pathways. 298 Currently used drugs against osteoporosis, hypercalcemia, Paget's disease, type II diabetes, depression, anxiety, and pancreatic diseases operate by modulating secretin GPCRs.
Five mutations or deletions in secretin family GPCRs were associated with human bone diseases or animal bone dysfunctions. A CALCR SNP was associated with BMD, bone mass, and fracture risk. [299][300][301][302][303] Multiple reports connected a Pro447Leu (rs1801197) polymorphism of CALCR and osteoporosis-related phenotypes and fracture risk in postmenopausal women, 299,[301][302][303][304][305][306] and an intronic SNP of rs2051748 was also significantly associated with vertebral trabecular BMD in older Caucasian men. 300 Zupan et al. found that there was a higher expression of CALCR in osteoarthritic patients. 299 Furthermore, Calcr +/− mice have a high bone mass with increased bone formation. 307 Rivadeneira et al. found that the rs9303521 SNP CRHR1 was associated with lumbar spine BMD in people of Northern European descent. 308 Several studies inferred that the GHRHR SNPs rs17159772, rs4988494, rs2267721, rs4988498, and rs4988505 were associated with reduced human height, indicating that GHRHR might affect normal human height variation. [309][310][311][312] Furthermore, the phenotype of pituitary dwarfism was also observed in individuals with  284 Wilson et al. 285 Naz et al. 286 Mouse 50% of male mice displayed abnormal claw morphology or lack of claws Frojmark et al. 284 Frizzled-9 Human The mutation was associated with reduced BMD Francke et al. 287 Wang et al. 273 Heilmann et al. 289 Mouse Induced osteopenia and reduced formation of new bone after fractures Albers et al. 288

SMOH Mouse
Reduced BMD, body length, and bone callus formation Cho et al. 38 Wang et al. 290 BMD bone mineral density, GPCR G protein-coupled receptor, SNP single-nucleotide polymorphism  266 Hoek et al. 267 GPR56 Human High levels were associated with rheumatoid factor and osteosarcoma proliferation and invasion Tseng et al. 260 Chen et al. 261 GPR110 Human Prognostic biomarker in osteosarcoma Liu et al. 262 GPR126 Human rs6570507, rs7774095, and rs7755109 SNPs were associated with AIS Qin et al. 250 Ikegawa et al. 248 Giampietro 251 Xu et al. 252 Kou et al. 253 Soranzo et al. 257 rs6570507, rs3748069, and rs4896582 SNPs were associated with reduced height Karnik et al. 254 Liu et al. 256 Soranzo et al. 257 The missense substitution (p.Val769Glu [c.2306T>A]) may be caused by severe arthrogryposis multiplex congenita Ravenscroft et al. 258 The rs536714306 SNP was associated with aggressive periodontitis Kitagaki et al. 259 Mouse Induced idiopathic scoliosis and pectus excavatum Karner et al. 265 GPR133 Human The rs1569019 and rs1976930 SNPs were associated with adult height Kim et al. 264 Kim et al. 249 Tonjes et al. 263 AIS adolescent idiopathic scoliosis, BMD bone mineral density, GPCR G protein-coupled receptor, SNP single-nucleotide polymorphism The role of GPCRs in bone diseases and dysfunctions J Luo et al.
and stimulation of phospholipase for PKC activation to stimulate downstream signaling events. 336 The 1-34 amino acid peptide of PTH (PTH(1-34)) is an anti-osteoporosis drug that functions by stimulating osteoblast proliferation, 337 increasing osteoblast activity, 338 and protecting osteoblasts from apoptosis 339 through direct binding to PTHR. 340 Interestingly, PTH(1-34) also maintains intervertebral disc homeostasis during aging, suggesting that PTH has the ability to maintain skeletal homeostasis 341 (Table 8).
Other 7TM receptors Several 7TM receptors did not fit into any family/group/cluster of the GRAFS classification system; therefore, these receptors are called other 7TM receptors. Most of them are orphan GPCRs. 46,47,268,275 There are five genes associated with bone diseases or dysfunctions in humans or mice from the other 7TM receptor group. GPR22 is an orphan GPCR. In silico and in vitro experiments suggested that the T-alleles of the rs3757713 and rs3815148 SNPs were associated with GPR22 expression in lymphoblasts. GPR22 was detected in cartilage and osteophytes in OA-induced mouse models but not in normal cartilage. Kerkhof et al. 346 identified SNP rs3815148 (located close to the GPR22 gene) as an OA susceptibility locus in a large association analysis of OA genetics with 14 938 OA cases and approximately 39 000 controls. Verleyen et al. found that altering the expression of Gpr22 in zebrafish embryos induced a downward-curving tail, which is often associated with defects in ciliogenesis. 347 GPR177, which is similar to the Frizzled family of GPCRs, is a Wnt signaling pathway component 348 involved in bone cell differentiation. As part of the RANK pathway, the gene positively regulates the NF-κB cascade. 349 Several multistage genome-wide association study meta-analyses identified four loci (rs1430742, rs2566755, rs2772300, and rs6588313 SNPs) in GPR177 that were associated with human lumbar spine, femoral neck, or total hip BMD. 308,350-353 Zhong et al. found that deletion of Gpr177 in mice resulted in bone loss, increased bone resorption, and defects in chondrogenesis and ossification 354,355 (Table 9).
The deletion of either Gpr30 41 or Gpr39 356 increased bone mass in mice, but in contrast, the deletion of Gpr40 43 or Gpr177 354 reduced mouse bone mass and BMD. GPR30, as an estrogen receptor, is activated by estrogen and the GPR30-specific agonist G1. 357 GPR30 activation elevates cAMP levels, intracellular Ca +2 mobilization, and transactivation of epidermal growth factor receptors. [358][359][360][361] GPR30 expression in human bone is limited to osteoblasts, osteocytes, and osteoclasts. 362 In immortalized rat skull preosteoblasts, Runx2 upregulated Gpr30 gene expression and increased osteoblast progenitor proliferation, suggesting that Gpr30 may promote osteoblast differentiation. 363 Confounding this, however, Ford et al. reported that Gpr30 loss increased bone mass, mineralization, and growth plate proliferation in male mice, 41 whereas Martensson et al. 364 reported that Gpr30 deletion reduced female mouse femur length.
Gpr39 is a zinc-sensing receptor that is expressed by osteoblast cell lines. 365 Zinc potently and specifically activates Gpr39 to induce Gq, G12/13, and Gs pathway signaling, suggesting that zinc is a physiologically important agonist. 366 Jovanovic et al. 356 found that Gpr39-deficient mice have higher bone stiffness and a higher mineral-to-matrix ratio, along with increased bone formation and osteoblast differentiation, suggesting that zinc sensing by Gpr39 is important in regulating collagen processing and mineralization, which are required for the proper maintenance of bone integrity.
GPR40 is highly expressed in pancreatic beta cells, where it interacts with medium-to-long chain fatty acids, [367][368][369] to potentiate glucose-induced insulin secretion. 370 GPR40 is also expressed in leukocytes, osteoclasts, and monocytes. 371,372 Cornish et al. 373 observed that a GPR40 agonist inhibits osteoclastogenesis, which is similar to the effects of free fatty acids. Furthermore, Gpr40 downregulation protects osteocytes from apoptosis. 374 Wauquier et al. 43 observed that Gpr40 −/− mice had a reduction in BMD and bone mass with higher promoting osteoclast differentiation, and Monfoulet et al. 375 observed a more severe OA-induced phenotype in Gpr40 −/− mice, marked by elevated tidemark exposure, osteophyte formation, and subchondral bone sclerosis (Table 9).

CONCLUSIONS
GPCRs play crucial roles in bone development, remodeling, and diseases by activating GPCR signaling pathways. Our results show that 92 receptors (5 glutamate family, 67 rhodopsin family, 5 adhesion, 4 frizzled/taste2 family, 5 secretin family, and 6 other 7TM reporters) were associated with bone diseases and dysfunctions (35 in humans and 72 in animals), and the catalog of diseases linked to GPCR malfunction continues to expand.
In summary, the GPCR superfamily plays a key role in regulating bone diseases and remodeling. Different GPCRs from different subfamilies may have similar physiological functions to regulate these processes; however, the same GPCR may have different physiological functions in different populations or animal models. Although the field has made significant progress in understanding

GPR177
Human Associated with reduced BMD Rivadeneira et al. 308 Deng et al. 350 Roshandel et al. 351 Styrkarsdottir et al. 352 Hsu et al. 353 Mouse Reduced bone mass and increased bone resorption Zhong et al. 354 Defects in chondrogenesis and ossification Zhong et al. 355 BMD bone mineral density, GPCR G protein-coupled receptor The role of GPCRs in bone diseases and dysfunctions J Luo et al.
how GPCRs influence bone development and diseases, much remains unknown. Since many GPCR mutations are embryonic lethal, the availability of mouse models to study GPCRs has been a significant barrier to progress. Fortunately, conditional knockout approaches have proven effective in many cases, allowing characterization of the detailed mechanisms involving GPCRs in bone diseases and dysfunctions. This should allow enormous advances in translational medicine, as GPCRs are generally regarded as a superb class of drug targets.