Effect of fluoride on 126 orphan GPCRs

We screened 126 GPCRs from class A, B, or C orphan GPCRs using β-arrestin recruitment assay. Relative luminescence values were determined from fluoride treated and untreated cells. Percentage RLU was calculated using the formula: %RLU= RLU from fluoride treated wells/ RLU from fluoride untreated wells (Table 4.1). Percentage RLU values more than 200 were considered significant. After fluoride treatment, out of the 126 screened human orphan GPCRs, only three GPCRs demonstrated percentage RLU >200. Additional information regarding the 3 orphan GPCRs demonstrating activation due to fluoride is outlined in Table 4.2.

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GPCR %RLU GPCR %RLU GPCR %RLU GPCR %RLU

GPR1 149.86 GPR111 258.89 GPRC5B 134.51 SUCN1 97.54 GPR3 109.16 GPR113 171.54 GPRC5C 90.80 TA1 92.27 GPR4 120.95 GPR114 144.20 GPRC5D 226.05 TAAR2 79.60 GPR6 93.34 GPR115 138.48 GPRC6A 113.50 TARR5 64.35 GPR12 72.79 GPR116 131.96 HCA1 162.89 TAAR6 115.58 GPR15 121.36 GPR119 95.70 HCA2 102.64 TARR8 81.74 GPR17 96.57 GPR123 128.30 HCA3 67.12 TAAR9 88.59 GPR19 141.49 GPR124 116.51 LPA4 125.11 CCR1 55.96 GPR20 142.03 GPR125 153.57 MAS1 125.03 CCR2 115.92 GPR26 96.77 GPR126 106.69 MAS1L 118.75 CCR3 95.46 GPR32 110.23 GPR132 109.92 MRGPRD 111.31 CCR4 109.65 GPR34 86.18 GPR133 128.13 MRGPRE 176.81 CCR5 117.28 GPR35 108.01 GPR141 87.06 MRGPRF 98.36 CCR6 109.41 GPR37 116.41 GPR142 320.78 MRGPRG 107.04 CCR7 70.12 GPR45 118.93 GPR143 121.50 MRGPRX1 79.86 CCR8 111.60 GPR50 134.19 GPR150 91.57 MRGPRX2 76.74 CCRL2 126.35 GPR52 92.00 GPR152 102.72 MRGPRX3 146.27 CD97 101.64 GPR55 118.14 GPR156 108.99 MRGPRX4 150.01 CMKLR1 90.49 GPR56 130.35 GPR157 98.02 RXFP1 76.87 CX3CR1 105.89 GPR61 130.76 GPR158 166.57 RXFP2 147.83 CXCR1 111.50 GPR63 133.15 GPR160 111.66 RXFP3 118.72 CXCR2 114.56 GPR64 154.80 GPR161 111.91 RXFP4 157.24 CXCR3 97.31 GPR65 120.57 GPR171 179.72 NPBW1 74.40 CXCR4 117.57 GPR75 130.21 GPR173 129.56 NPBW2 118.71 CXCR5 112.26 GPR78 107.35 GPR174 78.17 NPFF1 65.46 CXCR6 168.99 GPR82 115.84 GPR182 149.48 NPFF2 75.08 FFA1 105.18

GPR83 105.56 BBA3 97.41 OPN5 106.91 FFA2 103.09

GPR84 115.82 C5A 75.63 OXGR1 107.12 GPBA 110.75

GPR87 105.22 CCR10 130.98 PKR1 102.71 GPER 110.93

GPR92 116.29 ELD1 126.05 PKR2 70.67 GAL3 98.13

GPR97 97.55 FPR3 99.43 PRRP 76.54

GPR101 99.93 GPRC5A 187.08 QRFP 105.65

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HTL cells transfected with 126 human orphan GPCRs from class A, B, or C. Cells were treated with 0 or 1mM fluoride and relative luminescence units (RLU) were measured to calculate %RLU calculated using formula %RLU= RLU from fluoride treated wells/ RLU from fluoride untreated wells (IUPHAR class A database; IUPHAR class B database; IUPHAR class C database).

GPCR Class Human Chr. (AA) Mouse Chr. (AA) % RLU

GPR142 Class A 17q23 (462) 11 E2 (365) 321

GPR111 Class B 6p12.3 (642) 17 B3 (644) 259

GPRC5D Class C 12p13.3 (300) 6 G1 (345) 226

87 4.5 Discussion

GPCR’s forms the largest family and pharmacologically diverse group of mammalian membrane bound proteins. About 1/3rd of the known drugs target GPCR functions which suggest the importance of GPCRs in therapeutics. (Tang et al., 2012). GPCRs are of great interest due to its unique property to transduce extracellular signal into intracellular signaling events through heterotrimeric G proteins. GPCRS binds to a wide range of ligands including ions, small peptides, and organic compounds (Vardy and Roth, 2013). Phylogenetically human GPCRs are categorized in to five main families such as; Rhodopsin, Glutamate, Adhesion, Secretin, and Frizzled/Taste 2. Rhodopsin forms the largest family of GPCRs members of which are widely studied (Tang et al., 2012).

Deorphanization of the orphan GPCRs can be challenging due to lack of knowledge about the functionality and ligand specificity of these orphan receptors. (Chung et al., 2008). Part of the challenge arises due to that fact that most deorphanation assay involves detection of changes in the levels of second messengers (canonical pathway). However, it is important to understand that levels of the second messengers are regulated by the heterotrimeric G proteins and can be independent of GPCR of interest. Similarly lack of knowledge about intracellular signaling after orphan GPCR activation potentially limits the selection of proper positive and negative controls. The β-arrestin recruitment assay (non-canonical signaling) is independent of intracellular second messengers (Barnea et al., 2008), which distinguishes the β-arrestin recruitment assay from other assays that measure GPCR function.

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In the β-arrestin recruitment assay an orphan receptor is genetically modified to transduce an intracellular signal into a reporter gene transcription. Transcriptional activation of reporter gene provides a quantitative measure for GPCR activation. The reporter gene activity is independent of endogenous receptors and hence β-arrestin recruitment assay is sensitive (Allen et al., 2011).

Figure 4.5: Schematic of the β-arrestin recruitment assay

In the β-arrestin recruitment assay when a genetically modified GPCR (with attached transcription factor via protease site) binds to its ligand it leads to the recruitment of the arrestin-TEV protease fusion. Proteolytic cleavage at protease site liberates the tethered transcription factor (tTA). The tTA translocates to the nucleus leading to transcription of reporter gene. Increase in the reporter gene activity is used as a quantitative measure for receptor activation (Barnea et al., 2008).

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The β-arrestin recruitment assay monitors ligand binding-mediated receptor activation (non-canonical pathway). We screened human CASR and orphan GPCRs using this generality to identify receptor activation by fluoride through β-arrestin recruitment assay which provides a more quantitative assay, unaffected by endogenous receptor signaling (Tohgo et al., 2003).

Fluoride is known to activate various G-proteins, including the two regulatory proteins Gs and Gi of the AC system (Sternweis and Gilman, 1982). Fluoride mimics the effects of high calcium on several intracellular second messengers to mediate a time- and dose-dependent increase in the accumulation of IP3 in bovine parathyroid cells (Chen et al., 1988).

Fluoride does not influence β-arrestin recruitment by CASR in HTL cells which suggests that actions of fluoride on parathyroid gland to modulate PTH secretion (non-canonical pathway) are independent of CASR. Altering the calcium concentration did not have any impact on the fluoride-mediated arrestin recruitment which further supports our finding. There was a difference in β-arrestin recruitment based on difference in media calcium concentration. The role of media calcium concentration on CASR signaling has been previously documented (Roussanne et al., 2001). High media calcium increased arrestin recruitment and thereby luminescence readout. In our study 0mM calcium in the media demonstrated maximum arrestin recruitment which is in accordance with previously reported findings (Almaden et al., 2009; Nemeth et al., 1998). These findings suggests that increase in arrestin recruitment may be is responsible for a low extra-cellular calcium mediated increase in the PTH secretion (non-canonical pathway).

90

The IC50 concentration (1-2mM) determined in our experiments with R568 are comparable to those in previously documented study (Li et al., 2009). However Nemeth el al reported much lover IC50 values (~27nM) compared to our values (Nemeth et al., 1998). It is interesting to note that using arrestin recruitment assay in our study, R568 demonstrated an inverse agonist activity on CASR rather than positive allosteric modulator activity as previously described (Li et al., 2009; Nemeth et al., 2004; Wada et al., 2000). Our study demonstrated that the activity of R568 was specific for CASR and also extended to other known calcium sensing receptor such as GPRC6A.

Three orphan receptors identified by the β-arrestin assay: GPRC5D, GPR111 and GPR142 (Brauner-Osborne et al., 2001; Fredriksson et al., 2003), which were activated by fluoride treatment, are ubiquitously expressed. However, functional aspects of these three GPCRs are unknown. In a study by Everett et al to determine genetic basis for fluoride susceptibility in two strains of mice (A/J and 129P3/J) detected quantitative trait loci (QTLs) associated with dental fluorosis on chromosome 2 and 11(Everett et al., 2011). It is interesting to know that in mice GPR142 is localized on chromosome 11. Therefore, future studies will be directed to identify the role of GPR142, GPR111 and GPRC5D in fluoride mediated actions on hard tissues to understand the patho-physiology of dental and skeletal fluorosis.

The findings of the study can be summarized as follows: Fluoride can modulate PTH secretion independent of CASR activation. The possibility of involvement of other GPCRs in fluoride-mediated modulation of PTH cannot be rejected. Screening of the orphan GPCRs for fluoride-mediated receptor activation identified three

91

candidates: GPRC5D, GPR111, and GPR142. Future study of these candidates will help us to understand their role in the patho-physiology of dental and skeletal fluorosis and fluoride’s action on cellular biology.

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