bioRxiv preprint doi: https://doi.org/10.1101/760769; this version posted September 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

1

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3 The PAR2 signal prevents premature receptor cleavage and activation

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6 Belinda Liu1, Grace Lee1, Jiejun Wu, Janise Deming, Chester Kuei, Anthony Harrington, Lien Wang, 7 Jennifer Towne, Timothy Lovenberg, Changlu Liu*, Siquan Sun*

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10 Janssen Research & Development, LLC, 3210 Merryfield Row, San Diego, USA

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14 1 These authors contributed equally to this manuscript.

15 *Corresponding author

16 Email: [email protected]; [email protected]

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1 bioRxiv preprint doi: https://doi.org/10.1101/760769; this version posted September 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

18 Abstract

19 Unlike closely related GPCRs, protease-activated receptors (PAR1, PAR2, PAR3, and PAR4) have

20 a predicted signal peptide at their N-terminus, which is encoded by a separate exon, suggesting that the

21 signal of PARs may serve an important and unique function, specific for PARs. In this report, we

22 show that the PAR2 signal peptide, when fused to the N-terminus of IgG-Fc, effectively induced IgG-Fc

23 into culture medium, thus behaving like a classical signal peptide. The presence of PAR2 signal

24 peptide has a strong effect on PAR2 cell surface expression, as deletion of the signal peptide (PAR2∆SP)

25 led to dramatic reduction of the cell surface expression and decreased responses to trypsin or the synthetic

26 peptide ligand (SLIGKV). However, further deletion of the tethered ligand region (SLIGKV) at the N-

27 terminus rescued the expression and the response to the synthetic peptide ligand,

28 suggesting that the signal peptide of PAR2 may be involved in preventing PAR2 from intracellular protease

29 activation before reaching the cell surface. Supporting this hypothesis, an Arg36Ala mutation on PAR2∆SP,

30 which disabled the trypsin activation site, increased the receptor cell surface expression and the response

31 to ligand stimulation. Similar effects were observed when PAR2∆SP expressing cells were treated with

32 protease inhibitors. Our findings indicated that these is a role of the PAR2 signal peptide in preventing the

33 premature activation of PAR2 from intracellular protease cleavage before reaching the cells surface. The

34 same mechanism may also apply to PAR1, PAR3, and PAR4.

35

36 Introduction

37 G- coupled receptors (GPCRs) are a class of 7 transmembrane domain cell surface receptors

38 and consist of the largest receptor family in mammals and other organisms. They are involved in the signal

39 transduction of almost every system in human physiology, including the sensory, metabolic, endocrine,

40 immune, and the nervous systems. Unlike many other cell surface receptors that have a classical signal

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41 peptide to lead the to cells surface, the majority of GPCRs (>90%) do not have a signal peptide

42 (1). In general, class B receptors such as the receptor (2), corticotropin-releasing hormone (CRH)

43 receptors (3), the receptor (4), and Glucagon-like peptide receptors (5) and the class C GPCRs,

44 such as metabotropic glutamate receptors (6), GABA receptors (7), and adhesion GPCRs (8),which have

45 relatively large N-terminal extracellular domains are more likely to have signal peptides than class A

46 receptors (Fig 1A). It is hypothesized that the presence of the signal peptide helps the large hydrophilic N-

47 terminus to cross the plasma membrane. Most class A GPCRs do not have classical signal peptides. It is

48 believed that the first transmembrane domain of these class A GPCRs serves as a signal anchor sequence

49 allowing these receptors to translocate to the after translation and assembly in the

50 (ER) (9). Protease-activated receptors (PARs), including PAR1, PAR2, PAR3, and

51 PAR4 belong to class A GPCR receptor sub-family (10). Homology-wise, they are very closely related to

52 cysteinyl leukotriene receptors (CYSLT), niacin receptors (GPR109), lactic acid receptor (GPR81), and the

53 succinate receptor (GPR91). Unlike their closest neighbors (Fig 1B), which do not possess a signal peptide,

54 all PARs have a predicted signal peptide at their N-termini (Fig 1C). Genomic analyses show, in contrast

55 to their closest neighbors that are all encoded by single exon , PARs have an additional exon encoding

56 only the signal peptides (Fig 1C), suggesting that these signal peptides may play a specific role for PARs.

57 In this report, we used PAR2 to study the importance of the signal peptide in PAR receptor function and

58 localization.

59

60 Fig 1. PAR receptors are unique group of receptors in the Class A subfamily. A. Examples of GPCR subfamily 61 members and signal peptide possession. The signal peptide regions in Class B and C are shown in red. B. PAR 62 receptors and their closest neighbors, grouped by sequence similarity. C. The N-terminal sequence of 63 PAR1-4. The signal peptides are shown in blue. Each PAR receptor is encoded by 2 exons. The protein regions coded 64 by the first exons are underlined. The Arg residues involved in receptor cleavage and activation are shown in red. The 65 first transmembrane domains are shaded.

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67 Materials and methods

68 Reagents

69 The PAR2 agonist peptide ligand, SLIGKV, was synthesized by Innopep, Inc. (San Diego, CA).

70 Trypsin (sequencing grade), thrombin, and protease inhibitors were purchased from Sigma Aldrich (St.

71 Louis, MO).

72 Quantitative PCR analysis of the mRNA expression levels of PARs

73 Total RNAs were isolated from COS7, HEK293, and CHO-K1 cells respectively using a RNA

74 isolation kit (RNeasy Mini Kit) from Qiagen. cDNAs were synthesized from the isolated RNA using a

75 cDNA synthesis kit (Advantage RT-PCR kits) from Clontech (Mountain View, CA). Specific primers

76 designed according to human, monkey, and hamster PAR1, PAR2, PAR3, and PAR4 were used to quantify

77 each mRNA expression using a qPCR machine (QuantStudio, ABI) as described (11). In parallel, primers

78 for -actin were used to amplify -actin cDNA as the internal controls. The relative expressions of different

79 PAR mRNAs were normalized using the expression level of -actin. The qPCR primers were designed

80 based on the published cDNA sequences and the primer sequences are listed in Table 1.

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Gene Accession No Forward Primer Sequence Reverse Primer Sequence

Human PAR1 NM_001992.4 CCATTTTGGGAGGATGAGGAG AGGATGAACACAACGATGGCCAT

Human PAR2 NM_005242.5 ATGGCACATCCCACGTCACTGGA GAACCAGATGACAGAGAGGAGGTC

Human PAR3 NM_004101.3 ATGCTACCATGGGGTACCTGAC GTTGCCATAGAAGATGACTGTGGT

Human PAR4 NM_003950.3 CCTCCACCATGCTGCTGATGAA AGGTCTGCCGCTGCAGTGTCA

Human Actin NM_001101.4 GGTCATCACCATTGGCAATGAG GATCTTGATCTTCATTGTGCTG

Monkey PAR1 XM_011730122 CCATTTTGGGAGGATGAGGAG AGGATGAACACAACGATGGCCAT

Monkey PAR2 XM_011730121 ATGGCACATCCCACGTCACTGGA GAACCAGATGACAGAGAGGAGGTC

Monkey PAR3 XM_003899832 ATGCTACCATGGGGTACCTGAC GTTGCCATAGAAGATGACTGTGGT

Monkey PAR4 XM_011759280 CCTCCACCATGCTGCTGATGAA AGGTCTGCCGCTGCAGTGTCA

Monkey Actin NM_001033084 GGCACCACACCTTCTACAATG GGTCCAGACGCAGGATGGCAT

Hamster PAR1 XM_007636187 CGCCAGCCAGAATCTGAGATG CGAGGGGATGAAGAGCCTCAG

Hamster PAR2 XM_007632089 GGACGCAACGGTAAAGGAAGA CTTCGTCCGGAAAAGGAAGAC

Hamster PAR3 XM_003498712 CTTCTGCCAGCCACTTTTTGC GGAACTTCTCAGGTATCCCATGGT

Hamster PAR4 XM_007629105 GGGAAATTCTGTGCCAACGAC GGCCAATAGTAGGTCCGAAAC

Hamster Actin NM_001244575 GTAGCCATTCAGGCTGTGCTG ATGCAGCAGTGGCCATCTCCT

87 88 Table 1. The Genbank accession numbers for cDNA sequences used for PCR primer design and 89 primer sequences for qPCRs. All primer sequences are shown from 5’ to 3’. 90

91 Generation of PAR1, PAR2 knock-out cell line

92 A PAR1, PAR2 knock-out HEK293 cell line was created by Applied StemCells (Milpitas, CA)

93 using a CRISPR/Cas9 approach. Briefly, the coding region (nucleotide 374-643) of PAR1, which encodes

94 the protein region TM2 to TM3 of PAR1, was deleted. Similarly, the coding region (281-627) of PAR2,

95 which encodes the protein region TM2 to TM3 of PAR2, was deleted. Single cell clones were isolated. PCR

96 analysis of the genomic DNA by PCR followed by DNA sequencing were used to confirm the deletion of

97 the DNA fragments.

98

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99 Molecular cloning of PAR2 constructs

100 The PAR2 coding region was amplified by polymerase chain reaction (PCR) using primers (5’ atg

101 tct GAA TTC GCC ACC atg cgg agc ccc agc gcg gcg tgg ctg ctg -3’; reverse primer: 5’- atg tct GCG GCC

102 GCt caa tag gag gtc tta aca gtg gtt gaa ct-3’) designed based on the published PAR2 coding sequence

103 (Genbank Accession No. NM_005242.5). Human colon cDNA purchased from Clontech (Palo Alto, CA)

104 was used as the template. Expanded high fidelity PCR system (Roche Life Science, Indianapolis, IN) was

105 used to amplify the full length PAR2 cDNA coding region. The resulting DNA was digested using EcoR1

106 and Not1 restriction enzymes (Promega, Madison, WI) and then cloned into pcDNA3.1 (Invitrogen,

107 Carlsbad, CA). The insert region was then sequenced by Eton Biosciences (San Diego, CA) and the identity

108 of the entire coding region was confirmed.

109 Expression constructs for PAR2 with an Arg36Ala mutation (PAR2(R36A)), PAR2 without the

110 signal peptide (PAR2∆SP), PAR2∆SP with an Arg36Ala mutation (PAR2∆SP(R36A)), and PAR2 without

111 the signal peptide and the tethered ligand (PAR2∆SP∆L) were generated by site directed mutagenesis using

112 overlapping PCR approach (12).

113 Genes for PAR2 with the signal peptide coding regions replaced by the signal peptide, or

114 the signal peptide were synthesized by Eton Biosciences. Similarly, expression constructs

115 for various PAR2 variant with a GFP fused to the C-termini, human IgG-Fc coding region with or without

116 a PAR2 signal peptide coding region, with an insulin, or with an insulin receptor signal peptide coding

117 region were synthesized. The genes were cloned into pcDNA3.1 and the entire coding regions were

118 sequenced to confirm the identities.

119 Intracellular Ca2+ mobilization assay

120 FLIPR-Tetra (Molecular Device, San Jose, CA) was used to monitor intracellular Ca2+ mobilization

121 in HEK293 cells, HEK293 cells with PAR1 and PAR2 knocked-out, and cells transiently transfected with

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122 various PAR2 expression constructs. Cells were grown in 96-well polyD-lysine coated black FLIPR plates

o 123 (Corning) in DMEM supplemented with 10% FBS, 1 mM pyruvate, 20 mM HEPES, at 37 C with 5% CO2.

124 For transient transfection, cells were grown in 96-well polyD-lysine coated black FLIPR plates and

125 transfected using FuGENE HD (Promega, Madison, WI) as the transfection reagent according to the

126 manufacturer’s instructions. For samples treated with protease inhibitors, protease cocktail was added to

127 cell culture one day after transfection and incubated overnight. Two days after transfection, cell culture

128 media were removed, and cells were washed using HBSS buffer plus 20 mM HEPES. Ca2+ dye (Flura 3)

129 diluted in HBSS buffer plus 20 mM HEPES was used to incubate cells at RT for 40 min to allow Ca2+ to

130 enter cells. Intracellular Ca2+ mobilization stimulated by various concentrations of ligands (trypsin, or

131 peptide ligand) was monitored by FLIPR-Tetra as described (13). The untransfected cells were used as

132 negative controls.

133 Enzyme linked immunosorbent assay (ELISA) for the measurement of IgG-FC secretion

134 COS7 cells were grown in 6 well plates with DMEM supplemented with 10% FCS, 1 mM pyruvate,

o 135 20 mM HEPES, at 37 C with 5% CO2 and transfected by different expression constructs for human IgG-Fc

136 with various signal peptide coding regions using LipofectAmine (Invitrogen, Calsbad, CA) as the

137 transfection reagent according to the manufacturer’s instructions. Untransfected cells were used as negative

138 controls.

139 To measure the secreted human IgG-FC in the medium, one day after transfection, the cells were

140 washed 3 times using PBS and then cultured in serum free DMEM plus 1 mM pyruvate and 20 mM HEPES.

141 Three days after transfection, the conditioned media from the transfected cells were harvested and

142 centrifuged at 10,000 g at 4oC for 20 min to remove the cell debris. 50 ul of the conditioned medium from

143 each transfection was incubated in one well of a 96-well ELISA plate (UltraCruz® ELISA Plate, high

144 binding, 96 well, Flat bottom, Santa Cruz Biotechnology) at 37oC for 1 hr to allow protein in the media to

145 adsorb to the plates. The plates were washed 3 times using PBS + 0.1% Tween-20 (PBST) and blocked

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146 using 3% no-fat milk in PBST for 30 min at RT and then incubated using HRP-conjugated goat-anti-human

147 Ig-GF antibody (50 ng/ml) diluted in 3% no-fat milk in PBST at 4oC overnight. The plates were washed 3

148 times using PBST and then developed using an ELISA developing kit (BD Biosciences). The optical

149 densities at 450 nm were read using an ELISA plate reader (Molecular Devices).

150 To measure intracellular IgG-Fc protein, one day after transfection, cells were trypsinized and

151 seeded in 96-well culture plates (30,000 cell/well) and grown in DMEM supplemented with 10% FCS, 1

152 mM pyruvate, 20 mM HEPES. Three days after transfection, the media were removed, and cells were

153 washed using PBS, and then fixed by 10% formaldehyde in PBS at RT for 15 min. The cells were penetrated

154 using 1% Triton-X-100 at RT for 10 min and blocked using 3% no-fat milk in PBST for 30 min at RT. The

155 cells were then incubated using HRP-conjugated goat-anti-human IgG-Fc antibody and the plate was

156 developed and read as described above.

157 Immuno-fluorescent staining of intracellular IgG-Fc

158 COS7 cells were transfected with various IgG-Fc expression constructs. One day after transfection,

159 cells were trypsinized and seeded in a 4-well cell culture chamber slides (Stellar Scientific, Baltimore, MD)

160 (60,000 cells/well). Three days after transfection, the media were removed, and cells were washed using

161 PBS, and then fixed by 10% formaldehyde in PBS at RT for 15 min. The cells were penetrated using 1%

162 Triton-X-100 at RT for 10 min and blocked using 3% no-fat milk in PBST for 30 min at RT. The cells were

163 then incubated using FITC-labelled goat-anti-human IgG-FC antibody (ThermoFisher Scientific) (200

164 ng/ul) diluted in 3% no-fat milk in PBST at 4oC overnight. The slides were then washed 3 times using PBST

165 and then dried using cool air and viewed under a fluorescent microscope.

166 Identification of the signal peptide cleavage site of PAR2

167 COS7 cells were grown in 15 cm dishes in DMEM supplemented with 10% FCS, 1 mM pyruvate,

o 168 20 mM HEPES, at 37 C with 5% CO2 and transfected by the expression construct of human IgG-FC with

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169 the N-terminus of PAR2 using LipofecAmine. One day after transfection, the cells were washed 3 times

170 using PBS and then cultured in serum free Opti-MEM (Life Technology) plus Pen/Strep. Three days after

171 transfection, the media were collected and centrifuged to remove the cell debris. The supernatants were

172 passed through a Protein A (Sigma) affinity column. The column was washed with PBS and eluted using

173 0.1 M Glycine/HCl (pH 2.8) and then neutralized using 1 mM Tris-HCl, pH 8.0. The eluted protein was

174 first treated with PNGase-F (Promega) to remove the N-linked glycosylation and then analyzed by mass

175 spectrometry to determine the N-terminal sequence. Protein sequencing was performed using a generic in-

176 solution protein digestion and LC-MS/MS method. Briefly, a 10 ul protein sample in 50 mM ammonia

177 bicarbonate buffer (pH 7.8) was reduced by 11.3 mM dithiothreitol at 60oC for 30 min (without urea),

178 alkylated with 37.4 mM iodoacetamide (RT, 45 min), and then digested with 0.2 ug Trypsin (37oC,

179 overnight). LC/MS analysis was carried out on an Agilent 1290 UHPLC coupled to a 6550 qTOF mass

180 spectrometer, under the control of MassHunter software version 4.0. Chromatography was run with an

181 Agilent AdvanceBio Peptide Map column (2.1 x 100 mm, 2.7 m) using water/acetonitrile/0.1% formic

182 acid as mobile phases, and mass spectrometric data were acquired in both MS and MSMS modes.

183 Protease inhibitor treatment of cells recombinantly expressing PAR2 receptors

184 The wild type and various mutant PAR2 variant expression constructs were transiently transfected

185 into HEK293 cells with par1 & par2 genes knocked-out. 24 hrs after transfection, cells were treated for 12

186 hrs with a protease inhibitor cocktail including 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride

187 (AEBSF, 500 uM), Leupeptin (50 uM), aprotinin (50 uM).

188 Measurement of the total and cell surface expression of PAR2 by ELISA

189 ELISA was used to measure the total and cell surface PAR2 protein expression. The wild type and

190 different mutant PAR2 variants were transiently expressed in HEK293 cells with the endogenous PAR1

191 and PAR2 knocked-out. The cells were transfected in 10 cm cell culture dishes and, 24 hr after transfection,

192 split into 96-well polyD-lysine coated plate. 48 hr post transfection, cells were fixed as described above.

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193 To measure the total PAR2 expression, the fixed cells were penetrated using 1% triton-X-100, and blocked

194 with 3% no-fat milk, and then incubated with a monoclonal antibody [3 g/ml, mouse anti-human PAR2

195 (BioLegand, San Diego, CA)], which recognizes the N-terminal region (amino acid residues 37-62) of the

196 human PAR2, at 4oC overnight. The plate was washed with cold PBS 3 times and then incubated using a

197 HRP-conjugated goat-anti-mouse IgG secondary antibody (30 ng/ml, Pierce) at RT for 1 hr. The plate was

198 washed again using PBS and developed using an ELISA developing kit as described above. To measure

199 the cell surface PAR2 expression, the ELISA assays were performed in the same manner as the total PAR2

200 measurement without using triton-X-100 as the cell penetrating agent.

201 Measurement of the total expression and cellular localization of PAR2-GFP fusion proteins

202 GFP fusion proteins of PAR2 wild type and various mutants were transiently expressed in 96-well

203 poly-D-lysine plates in HEK293 cells with the endogenous PAR1 and PAR2 knocked-out as described

204 above in methods for Intracellular Ca2+ mobilization assay. 48 hrs after transfection, the media were

205 aspired, and cells were fixed using 4% Paraformaldehyde in PBS (Sigma). The fluorescent intensities of

206 the cells were read using an Envision plate reader (PerkinElmer). The fixed cells were then analyzed using

207 a confocal microscopy for PAR2 cellular localizations.

208

209 Results and discussions

210 PAR2 signal peptide behaves as a classical signal peptide

211 PAR2 signal peptide leads IgG-Fc fragment secretion to the medium

212 A classical signal peptide is typically found at the N-termini of either secreted proteins (such as

213 insulin) or cell surface proteins (such as insulin receptor). It typically consists of a stretch of 20-30

214 hydrophobic amino acid residues. Its known function is to help a secreted or a cell surface protein to target

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215 the ER during protein translation and cross the plasma membrane. PAR2 has a predicted signal peptide

216 sequence at its N-terminus and we hypothesized that it may function as a classical signal peptide. To address

217 this, we devised a few expression constructs (Fig 2A) to test whether the signal peptide of PAR2 enables

218 the secretion of human IgG-Fc fragment (which lacks a signal peptide) into the cell culture medium. IgG-

219 Fc was used since it was easy to detect using ELISA or immune-staining. When recombinantly expressed

220 in mammalian cells, without a signal peptide, IgG-Fc is only expressed intracellularly. In contrast, with a

221 signal peptide, IgG-Fc can be secreted into the cells culture medium. One IgG-Fc construct contained the

222 N-terminus of PAR2 with its signal peptide and another with the PAR2 N-terminus in which its signal

223 peptide was deleted. As positive controls, constructs with an insulin signal peptide (a secreted protein signal

224 peptide) or an insulin receptor signal peptide (a cell surface receptor signal peptide) fused to human IgG-

225 Fc were also used in the experiment. We used immuno-staining (Fig 2B) and ELISA (Fig 2C) to detect and

226 measure IgG-Fc expression in the transfected cells and demonstrated that all cells transfected with various

227 IgG-Fc expression constructs expressed IgG-Fc in the cells. Consistent with our hypothesis, our results

228 showed that fusing the N-terminus of PAR2 with its signal peptide to the human IgG-Fc, effectively led to

229 the secretion of IgG-Fc to the medium, thus functioning similarly to that of the insulin signal peptide or the

230 insulin receptor signal peptide (Fig 2D). In contrast, fusing the PAR2 N-terminus without its signal peptide

231 failed to lead to the secretion of IgG-Fc into the medium.

232

233 Fig 2. PAR2 signal peptide behaves like a classical signal peptide. A. Expression constructs for testing the roles of the signal 234 peptide of PAR2 in leading IgG-Fc secretion. The N-terminus of PAR2 with its signal peptide (PAR2), the N-terminus of PAR2 235 without the signal peptide (PAR2∆SP), the N-terminus of insulin (IN), and the N-terminus of insulin receptor (IR) are fused to the 236 human IgG-Fc fragment respectively. The signal peptide regions of PAR2, insulin, and insulin receptor are highlighted in red and 237 underlined. Human IgG-Fc fragment is highlighted in green. B, C. Detection of IgG-Fc expression in cells by immuno-fluorescent 238 staining and ELISA. COS7 cells expressing various IgG-Fc fusion proteins as indicated were fixed, penetrated using detergent, and 239 then detected or stained by FTIC-labeled fluorescent antibodies (B) or by ELISA (C). D. Detection of IgG-Fc secretion into media 240 by ELISA. Serum free conditioned medium from COS7 cells expressing various IgG-Fc fusion proteins with different N-termini, 241 including PAR2 N-terminus (PAR2), PAR2 N-terminus without the signal peptide (PAR2∆SP), the N-terminus of insulin (IN), 242 and the N-terminus of insulin receptor (IR). Untransfected cells were used as the negative control (NC). The experiments have 243 been performed 3 times and very similar results have been observed. Example data is shown. 244

245

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246 PAR2 signal peptide is cleaved from the mature protein

247 It has been reported that for the CRF2(a) receptor, the signal peptide may not be cleaved from the

248 mature proteins following membrane insertion (14). To determine if this is the case for the PAR2 signal

249 peptide, we examined whether the signal peptide of PAR2 is cleaved from the mature IgG-Fc protein with

250 the PAR2 N-terminus following secretion. The conditioned medium from the COS7 cells transfected with

251 the expression construct for PAR2 N-terminus fused to IgG-Fc (Fig 2A) was collected. Secreted PAR2-

252 IgG-Fc fusion protein was affinity purified, glycosylation moieties removed, and then analyzed by mass

253 spectrometric (MS) protein sequencing. Our results showed that the most N-terminal sequence that matches

254 PAR2 sequence is TIQGTNR (Fig 3), suggesting that the signal peptide has been cleaved following the

255 protein secretion, with the cleavage site being between residues Gly24 and Thr25. Interestingly, a variant

256 sequence TIQGTDR was also observed. This sequence differs from TIQGTNR by one residue (from N to

257 D). Since the residue Asn30 is a part of a NRS sequence (a N-linked glycosylation site, Fig 3) and

258 glycosylated Asn residues are converted to Asp after de-glycosylation by PNGase-F, our results suggest

259 that at least part of the expressed protein is glycosylated at this N-linked glycosylation site.

260 Fig 3. Determination of the N-terminal sequence of PAR2 mature protein. The N-terminal extracellular region of PAR2 is 261 fused to the N-terminus of IgG-Fc. The predicted signal peptide of PAR2 is shown in red and underlined. The IgG-Fc region is 262 shown in green. The potential N-linked glycosylation site, NRS, is underlined. The protein is expressed in COS7 cells and affinity 263 purified. The N-terminus of the purified protein was determined by MS sequencing after trypsin digestion. Two sequences have 264 been observed: TIQTNR and TIQTDR representing unglycosylated and glycosylated PAR2 N-termini. 265

266 PAR2 signal peptide is important for PAR2 receptor functional expression and

267 activation by its ligands

268 Generation of a PAR1 & PAR2 knock-out HEK293 cell line for recombinant expression and

269 characterization of the PAR2 receptor

270 To evaluate the receptor localization and function of recombinant PAR2, it is essential to have a

271 host mammalian cell line that does not express endogenous PAR2 or other PAR receptors. We tested a few

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272 commonly used mammalian cells for recombinant expression, including HEK293, CHO-K1, and COS7

273 cells and found that all three cell lines express relatively high PAR1 and PAR2 mRNA (Fig 4A). In addition,

274 in functional assays, they all responded to PAR1 and PAR2 ligands (thrombin and trypsin, respectively)

275 (Fig 4B-D). Since the presence of naturally expressed PAR1 and PAR2 in these host cells would complicate

276 the characterization of the recombinantly expressed PAR2, we created a HEK293 cell line (which does not

277 express PAR3 and PAR4) with both par1 and par2 genes knocked-out by CRISPR/cas9 (Fig 4E).

278 Pharmacological characterization of this cells line demonstrated that the loss of both par1 & par2 led to a

279 lack of responses to PAR1 ligand (thrombin) or PAR2 ligand (trypsin) stimulation (Fig 4F). These cells

280 were then used to study expression and localization of recombinant PAR2.

281 Fig 4. CHO-K1, COS7, and HEK293 cells express PAR1 and PAR2 receptors. A. CHO-K1, COS7, and HEK293 cells naturally 282 express high levels of PAR1 and PAR2 mRNA but express little or no PAR3 and PAR4 mRNA. qPCR analysis was used to quantify 283 the mRNA expression. Specific primers for each of PAR1, PAR2, PAR3, and PAR4, were used to quantify the respective mRNA 284 expression using cDNA made from each cell line as the templates. -actin primers were used to quantify -actin mRNA expression 285 as the internal controls. The relative mRNA expression of PAR1, PAR2, PAR3, and PAR4 were first normalized using -actin 286 expression, and then normalized using the PAR1 expression level in CHO-K1 cells, which is arbitrarily set as 100%. The relative 287 expressions of other genes are represented as percentage of PAR1 mRNA level in CHO-K1 cells. The results shown are mean ± sd 288 (n = 3). B, C, D. CHO-K1, COS7, and HEK293 cells naturally express PAR1 and PAR2 receptors and respond to thrombin (PAR1 289 ligand) and trypsin (PAR2 ligand) stimulations. FLIPR assays were used measure receptor activation as indicated by intracellular 290 Ca2+ mobilization. Relative fluorescent units (RFU) are the readout for fluorescent intensities for Ca2+ mobilization signals. Various 291 concentration of thrombin or trypsin were used as the ligands to activation the receptors. The assays were performed in triplicates 292 at each data point and mean ± sd are shown. E. Sequencing analysis of the genomic DNA from par1 & par2 knock out HEK293 293 cells. The results show that a 270 bp deletion in par1 and a 347 bp deletion in par2 gene have been achieved. The deletions 294 removed the coding regions from TM2 to TM3 for both PAR1 and PAR2 proteins. The vertical lines indicate the deletion sites. F. 295 Characterization of par1 & par2 knock-out HEK293 cells. FLIPR assays were used to characterize receptor activation as indicated. 296 Wild type HEK293 cells were used as the positive control. The assays were performed in triplicates at each data point and mean ± 297 sd are shown.

298

299 Deletion of the signal peptide reduced the functional expression of PAR2, which can be

300 rescued by a replacement signal peptide

301 To assess the functional role of the PAR2 signal peptide, we made a few modifications to the PAR2

302 N-terminus, including a N-terminal deletion to remove the signal peptide (PAR2∆SP) and the replacement

303 of the PAR2 signal peptide with an insulin signal peptide (PAR2-INSP), or an insulin receptor signal

304 peptide (PAR2-IRSP) (Fig 5A). Pharmacological characterization of the modified receptors using FLIPR

305 assay (Fig 5B, C) showed that the recombinantly expressed PAR2 responds to trypsin (EC50 = 1.5 nM) and

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306 PAR2 agonist peptide (PAR2-AP) (EC50 = 50 nM) with much higher sensitivity compared to the

307 endogenously expressed PAR2 in HEK293 cells (EC50 = 10 nM for trypsin and EC50 = 1.5 uM for PAR2-

308 AP). This is likely due to the over-expression of the recombinant receptor causing a super-pharmacology

309 phenomenon (15). In this case, the EC50 value is a good indicator of the relative number of receptors at the

310 cell surface. Compared to the cells expressing the wild type PAR2, cells expressing PAR2 without its signal

311 peptide demonstrated dramatically reduced sensitivity to both trypsin and PAR2-AP (EC50 for trypsin: 50

312 nM; EC50 for PAR2-AP: 5.8 uM), suggesting the signal peptide is an important component of PAR2

313 functional cell surface expression. Supporting this hypothesis, replacement of the PAR2 signal peptide

314 either with a signal peptide from insulin or from the insulin receptor fully restored the receptor ligand

315 sensitivity (Fig 5).

316 Fig 5. A signal peptide is important for functional expression of PAR2. A. schematic diagram showing the modifications to 317 PAR2 receptor. The N-terminal extracellular sequences of various PAR2 mutants are shown. Human PAR2 wild type (PAR2), 318 PAR2 with the signal peptide deleted (PAR2∆SP), PAR2 with an insulin signal peptide (PAR2-INSP) and an insulin receptor signal 319 peptide (PAR2-IRSP). The native signal peptide of PAR2 is shown in blue. The insulin and insulin signal peptides are shown in 320 green and purple respectively. The tether ligand sequence of PAR2 (SLIGKV) is highlighted in yellow. B, C. Characterization of 321 PAR2 mutants in FLIPR assay using trypsin or the synthetic PAR2 agonist peptide (PAR2-AP) as the ligands. Expression constructs 322 for PAR2 wild type receptor and various modifications were cloned into pcDNA3.1 and transiently expressed in HEK293 cells 323 with par1 and par2 knocked-out. Various concentrations of trypsin (B) or PAR-AP (C) were added to stimulate the intracellular 324 Ca2+ mobilization. Relative fluorescent intensity units (RFU) are shown. The experiments were performed in triplicates at each 325 data point and the results shown are mean ± sd. HEK293 cells with par1 and par2 genes knocked-out were used as the host cells 326 for recombinant expression of various PAR2 receptors. Untransfected cells were used as the negative controls (NC). The 327 experiments have been performed 3 times and very similar results have been observed. Example data is shown. 328

329 The presence of the tethered ligand is the reason that PAR2 needs the signal

330 peptide

331 Further deletion of the tethered ligand region rescues the functional expression of PAR2

332 without the signal peptide

333 PARs are activated by proteases, which generate new N-termini and expose the tethered peptide

334 ligands present in the N-terminal extracellular regions of the receptors. This unique receptor activation

335 mechanism, combined with the fact that signal peptide-less PAR2 has a poor response to ligand stimulation,

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336 led us to speculate that the necessity of the signal peptide for PAR2 may be related to the presence of

337 tethered ligand. We made a signal peptide-less PAR2 mutant with a further deletion to the region of the

338 tethered ligand (PAR2∆SP∆L) (Fig 6A). This mutant receptor lacks the signal peptide and the tethered

339 ligand sequence (SLIGKV) and is not activated by trypsin, however it can be fully activated by the synthetic

340 agonist peptide PAR2-AP similarly to the wild type PAR2 receptor in the FLIPR assay (Fig 6B). This

341 suggests that further deletion of the tethered ligand sequence (SLIGKV) restored functional cell surface

342 expression of PAR2 without the signal peptide. The results also suggest that, without a signal peptide,

343 PAR2 may be susceptible to unintended intracellular protease activation, leading to poor functional cell

344 surface expression.

345 Fig 6. Further deletion of the tethered ligand rescues the functional expression of PAR2 without the signal peptide. A. 346 schematic diagram showing the modifications to PAR2 receptor. The N-terminal extracellular sequences of various PAR2 mutants 347 are shown. Human PAR2 wild type (PAR2), PAR2 with the signal peptide deleted (PAR2∆SP), PAR2 with the signal peptide 348 deletion and with further deletion to the tether ligand region (PAR2∆SP∆L). The signal peptide of PAR2 is shown in blue. The 349 tether ligand sequence of PAR2 (SLIGKV) is highlighted in yellow. B, C. Characterization of mutant PAR2 receptors using FLIPR 350 assays. Various PAR2 expression constructs were transiently expressed in HEK293 with par1 and par2 knocked-out. Trypsin (B) 351 or the synthetic agonist peptide PAR2 ligand (PAR2-AP) (C) were used as the ligand to stimulate receptor activation. HEK293 352 cells with par1 and par2 genes knocked-out were used as the host cells for recombinant expression of various PAR2 receptors. 353 Untransfected cells were used as the negative controls (NC). The experiments were performed in triplicates at each data points and 354 the results shown are mean ± sd. The experiments have been performed 3 times and very similar results have been observed. 355 Example data is shown.

356

357 Mutation of Arg36 to Ala, which blocks the trypsin activation site, increased the functional

358 expression of PAR2 without the signal peptide

359 Trypsin activates PAR2 by cleaving the protein after residue Arg36. This process generates a new

360 N-terminus (with sequence SLIGKV---), which serves as a tethered ligand to activate the receptor.

361 Mutating Arg36 to Ala prevents the trypsin cleavage at this position, and therefore blocks trypsin-

362 mediated receptor activation. We made a mutation at the Arg36 position on PAR2 without the

363 signal peptide (PAR2∆SP(R36A)) and tested whether this mutation changes the level of functional

364 receptor expression. In parallel, we also made the same mutation on the full length PAR2 receptor

365 (PAR2(R36A)) and characterized these receptors in FLIPR assays after stimulation with trypsin

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366 or PAR2-AP. Our results demonstrated that the Arg36Ala mutation blocked, as expected, trypsin

367 activation of PAR2 without the signal peptide (Fig 7A). However, when the PAR2-AP was used

368 as the ligand, the mutant receptor (PAR2∆SP(R36A)) demonstrated a much higher sensitivity to

369 PAR2-AP compared to that of PAR2∆SP (Fig 7B). As a control, we made the same mutation in

370 full length PAR2 receptor (PAR2(R36A)), which responded to trypsin stimulation very poorly

371 (Fig 7B), responded to PAR2-AP stimulation almost identically to the full length PAR2 receptor

372 (Fig 7C). The small but detectable activation of PAR2(R36A) by trypsin (Fig 7B) could be due to

373 the cleavage of PAR2 by trypsin at Arg31, or Lys34 positions, resulting in tethered ligands with

374 poor activity for receptor activation.

375 Fig 7. Arg36 to Ala mutation helps the functional expression of PAR2 without a signal peptide. A. schematic diagram showing 376 the modifications/mutations to PAR2 receptor. The N-terminal extracellular sequences of various PAR2 mutants are shown. PAR2 377 wild type (PAR2), PAR2 with a Arg36 mutation to Ala (PAR2(R36A)), PAR2 with the signal peptide deleted (PAR2∆SP), PAR2 378 with the signal peptide deletion and with an Arg36Ala mutation (PAR2∆SP(R36A)) were used for characterizations. The signal 379 peptide of PAR2 is shown in blue. The tether ligand sequence of PAR2 (SLIGKV) is highlighted in yellow. The mutation Ala 380 residue from Arg36, which is involved in trypsin cleavage/activation of PAR2, is highlighted in red. The mutant receptors were 381 characterized in FLIPR assays using either trypsin (B) or PAR2-AP (C) as ligands. HEK293 cells with par1 and par2 genes 382 knocked-out were used as the host cells for recombinant expression. Untransfected cells were used as the negative controls (NC). 383 The experiments were performed in triplicates at each data points and the results shown are mean ± sd. The experiments have been 384 performed 3 times and very similar results have been observed. Example data is shown. 385

386 Protease inhibitor treatment increased functional expression of PAR2 without the signal

387 peptide

388 We hypothesized that serine protease inhibitors can help the functional expression of PAR2 without

389 a signal peptide by blocking premature intracellular protease-mediated activation. A protease cocktail

390 including AEBSF, Leupeptin, and aprotinin was used to inhibit ER and Golgi proteases (16, 17). Cells

391 expressing the wild type PAR2 and various mutant forms of PAR2 were treated with the protease inhibitor

392 cocktail and then tested for their responses to PAR2-AP stimulations. Trypsin was not used in this assay

393 because it is inhibited by the protease inhibitor cocktails. Our results showed that while protease inhibitors

394 did not affect the EC50 values of PAR2-AP stimulated responses for PAR2 wild type, PAR2(R36A),

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395 PAR2∆SP(R36A), and PAR2∆SP∆L, the protease cocktail clearly increased functional expression of

396 PAR2∆SP by decreasing the EC50 value (from 5.8 uM to 0.7 uM) (Fig 8).

397 Fig 8. Serine protease inhibitor cocktail increases the functional expression of PAR2 without the signal peptide. HEK293 cells with 398 par1 and par2 knocked out were used for the transient expression of various PAR2 proteins. Treatment with proteases inhibitor 399 cocktail (PI) lowered the Emax values for all receptors with similar degrees. Protease treated samples showed about 80% response 400 in Emax values compared with those of untreated cells. For good comparison of the EC50 values between samples treated and 401 untreated with the protease inhibitor cocktails, the results were normalized using their Emax values and the data were expressed as 402 the percentages of the Emax. The experiments were performed in triplicates at each data points and the results shown are mean ± 403 sd. The experiments have been performed 3 times and very similar results have been observed. Example data is shown.

404

405 Arg36Ala mutation and the protease inhibitor treatment increase the cell surface expression

406 of signal peptide-less PAR2

407 To confirm whether the reduced responses of signal peptide-less PAR2 to the ligand

408 stimulation is due to a lack of total receptor protein expression, and/or a lack of cell surface

409 expression, we used a monoclonal antibody against the amino acid residues 37-62 of PAR2 in

410 ELISA assays to measure the total and cell surface expression of the various forms of PAR2 and

411 effect of protease inhibitor treatment. We observed that PAR2 wild type and PAR2(R36A) mutants

412 have the highest total and cell surface protein expression as measured by ELISA. PAR2∆SP∆L

413 has slightly lower expression compared to that of the PAR2 wild type in both total and cell surface

414 expression. As this variant of PAR2 is missing amino acid residues 1-42, its reduced detection of

415 proteins expression could be due to the poor antibody recognition. PAR2∆SP(R36A) has further

416 lower total and cell surface expression and PAR2∆SP has the lowest total and particularly cell

417 surface expression levels (Fig 9). The data showed that the great majority of PAR2∆SP protein are

418 located intracellularly and only a small portion of it are present on the cell surface. For PAR2 wild

419 type, PAR2(R36A), and PAR2∆SP∆L, over 90% of the proteins are presented on the cell surface.

420 Corroborating the functional assays, protease inhibitor treatments increased the total and especially

421 the cell surface expression levels for PAR2∆SP while having little or no effects on the protein

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422 expression of other forms of PAR2 proteins (Fig 9). Stimulation of receptors using PAR2 peptide

423 ligand (PAR2-AP) decreased the cell surface and the total protein expression levels for all variants

424 of PAR2 except PAR2∆SP. This is likely due to that the majority of PAR2∆SP being intracellular,

425 and the ligand stimulation of the cell surface receptor, causing the subsequent internalization and

426 degradation of the stimulated receptors applies less to PAR2∆SP.

427 Fig 9. Cell surface and total protein expression of PAR2 wild type and mutants. HEK293 cells with par1 and par2 knocked- 428 out were used for the transient expression of various PAR2 proteins. PAR2 peptide ligand, PAR2-AP and protease inhibitor 429 cocktails (PI) were used for treatments. Medium was used as the control treatment. ELISA with or without cell penetrating reagent 430 was used to measure the total and cell surface and protein expression. The experiments were performed in triplicates at each data 431 points and the results shown are mean ± sd. The experiments have been performed 3 times and very similar results have been 432 observed. Example data is shown.

433

434 In parallel, to further facilitate the measurements of the protein expression and visualization

435 of protein cellular localizations, we constructed various PAR2 expression vectors by fusing a GFP

436 tag to the C-termini of the PAR2 wild type protein and the various PAR2 mutants (Fig 10A) and

437 expressed them in the par1 and par2 null HEK293 cell line. We measured the total expression

438 levels of PAR2 and the mutant proteins by measuring GFP fluorescence intensity of the various

439 GFP fusion proteins. In general, the results are very similar to that shown by ELISA using the anti-

440 PAR2 antibody except for PAR2∆SP∆L, which showed lower levels compared to that of PAR2

441 wild type in the ELISA assays, but showed similar expression levels to that of PAR2 wild type in

442 GFP intensities (Fig 10B). This result supports our earlier speculation that the reduced detection

443 of PAR2∆SP∆L expression is likely due to the poor recognition of PAR2∆SP∆L (which misses

444 the amino acid residues 37-42 of the recognition site) by the antibody. To investigate the cellular

445 localizations of PAR2 protein and its variants, we used confocal microscopy to analyze the cells

446 that express various PAR2 proteins at various conditions including the treatments with PAR2

447 ligand or protease inhibitors. PAR2 wild type, PAR2(R36A), and PAR2∆SP∆L proteins are

448 mainly localized on the plasma membranes (Fig 10C). PAR2∆SP can only be found intracellularly

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449 and little or none is on the plasma membranes, which is very similar to PAR2 wild type receptor

450 stimulated by the peptide ligand (PAR2+PAR2-AP, Fig 10C). For PAR2∆SP(R36A), a good

451 portion of protein is expressed on the plasma membrane and a large amount of protein can also be

452 found intracellularly. Interestingly, protease inhibitor treatment enabled the plasma membrane

453 expression of PAR2∆SP (PAR2∆SP+PI, Fig 10C). Overall, the observed GFP-tagged protein

454 cellular distribution is in agreement with the ELISA data (Fig 9). Interestingly, the cells expressing

455 PAR2∆SP(R36A) and protease inhibitor-treated cells expressing PAR2∆SP appeared to belong

456 two subcategories. One population of cells have good PAR2 plasma membrane localization,

457 mimicking the wild type PAR2, and another population of cells only have intracellular PAR2

458 which is similar to that of PAR2∆SP without the protease inhibitor treatment. Arg36Ala mutation

459 and the protease inhibitor cocktails (used in the assays) mainly block PAR2 cleavage/activation

460 by the serine proteases. However, cells may express other proteases that can cleave and activate

461 PAR2∆SP intracellularly but would not be blocked by the mutation or the protease inhibitor

462 treatment. It is possible that cells express different proteases under different conditions such as

463 different cell cycle stages (18-25).

464 Fig 10. Arg36Ala mutation and protease inhibitors increase the cell surface expression of PAR2-GFP without a signal 465 peptide. A. Schematic presentation of various PAR2-GFP fusion protein expression constructs. B. The expression levels of 466 various PAR2-GFP proteins with treatments of PAR2-AP, or protease inhibitors. Various PAR2-GFP expression constructs 467 were transiently expressed in HEK293 cells with par1 & par2 knocked-out. The transfected cells were treated either with medium 468 (medium), peptide agonist (PAR2-AP), or a protease inhibitor cocktails (PI), and the fluorescent intensities of the cells expressing 469 the PAR2-GFP fusion proteins were measured. Assays were performed in quadruplicates at each data point. C. Fluorescent images 470 from confocal microscope showing the cellular distributions of various PAR2-GFP fusion proteins under the treatments of 471 PAR2-AP or protease inhibitors. Untransfected cells were used as the negative control (NC). The fluorescent intensities shown 472 in the images are automatically adjusted for better viewing of the protein cellular distributions. The experiments have been 473 performed 3 times and very similar results have been observed. Example data is shown. 474

475 GPCRs are synthesized in the endoplasmic reticulum (ER) and transported to the Golgi

476 apparatus and then to the plasma membrane. There are many proteases present in the endoplasmic

477 reticulum and (16, 26-29) which may cleave the protease-sensitive PAR2

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478 activation site at Arg36 position during the protein synthesis and transportation process. This would

479 cause unintended or premature receptor activation, which would subsequently lead to receptor

480 internalization and degradation. The signal peptide of PAR2 is important for its functional

481 expression. However, the removal of the tethered ligand or the blockage of the receptor activation

482 by proteases dismissed the necessity of the signal peptide, suggesting that the signal peptide may

483 help prevent this unintended cleavage of PAR2 at the activation site during the protein synthesis

484 and/or transportation process. For cell surface proteins using signal peptides, their translocation to

485 ER and eventually the plasma membrane is mediated by ER (30, 31), which play roles

486 in protein compartmentalization (32-36) and segregation (31, 37, 38). Although the mechanism

487 remains unclear, we speculate that ER translocons may play the role in protecting PAR2 from

488 protease cleavage (Fig 11).

489 Fig 11. Schematic diagram showing the proposed role of PAR2 signal peptide in protecting PAR2 from protease cleavage 490 before reaching the plasma membrane. Without the signal peptide, the protease activation site of PAR2 is susceptible to protease 491 cleavage in ER and Golgi, leading to PAR2 activation before reaching the cell surface and subsequent translocation to lysosome 492 for degradation. With the signal peptide, PAR2 is bound by the signal peptide related complex and segregated/protected 493 from the cleavage by ER/Golgi proteases, allowing the receptor to reach the plasma membrane for sensing the extracellular trypsin 494 activation. The signal peptide of PAR2 at the N-terminus is shown in red. 495

496 The classical signal peptide has been known to help secreted proteins and cell surface

497 proteins to cross or become embedded in the cell membranes. In this report, through studying the

498 signal peptide of PAR2, we observed a function of the signal peptide to serve as a protector of

499 PAR2 from intracellular protease activation. Based on the high sequence similarities among the

500 PARs and their similar activation mechanisms, the signal peptides for PAR1, PAR3, and PAR4

501 may also serve the same function as that of PAR2. Cleavage of PARs by intracellular proteases

502 leads to the unintended activation of the receptors and the loss of function to sense the extracellular

503 signals. Therefore, with the protease-protection function, the signal peptides are critical for the

504 functions of the PAR family of receptors.

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505 Summary

506 To summarize, in this report we observed that deletion of the signal peptide of PAR2

507 decreased PAR2 cell surface expression with the most receptors accumulating intracellularly.

508 However, further deletion of the tether ligand of PAR2, which disables the activation of PAR2 by

509 trypsin, restored the receptor cell surface expression, suggesting that the necessity of the signal

510 peptide for PAR2 is related to the presence of the tether ligand sequence and the protease activation

511 mechanism. We hypothesize that the signal peptide of PAR2 protects PAR2 from intracellular

512 protease cleavage and activation. Without the signal peptide, PAR2 can be cleaved and activated

513 by intracellular proteases in the endoplasmic reticulum or Golgi apparatus, leading to the

514 unintended, premature receptor activation and resulting in intracellular accumulation. Supporting

515 this hypothesis, an Arg36Ala mutation at the trypsin activation site as well as protease inhibitor

516 treatments both increased the cell surface expression of the signal peptide-less PAR2 and

517 functional responses to ligand stimulation. Our results extend the knowledge of PAR2

518 expression/function and reveal a role of the signal peptide in protecting cells surface protein, and

519 perhaps the secreted proteins as well, from intracellular protease cleavages.

520

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23 bioRxiv preprint doi: https://doi.org/10.1101/760769; this version posted September 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/760769; this version posted September 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/760769; this version posted September 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/760769; this version posted September 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/760769; this version posted September 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/760769; this version posted September 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/760769; this version posted September 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/760769; this version posted September 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/760769; this version posted September 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/760769; this version posted September 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/760769; this version posted September 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.