Supporting Information. Post translational Modifications of Serotonin Type 4 Receptor Heterologously Expressed in. Mouse Rod Cells

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1 Supporting Information Post translational Modifications of Serotonin Type 4 Receptor Heterologously Expressed in Mouse Rod Cells David Salom,, Benlian Wang,, Zhiqian Dong, Wenyu Sun, Pius Padayatti, Steven Jordan #, John A. Salon # and Krzysztof Palczewski, * Polgenix Inc., Cleveland, Ohio 44106, USA; Department of Pharmacology and Center for Proteomics and Bioinformatics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, USA; and # Department of Molecular Structure, Amgen Incorporated, Thousand Oaks, CA , USA. These authors contributed equally to this work. * To whom correspondence should be addressed: Krzysztof Palczewski, Ph.D. Phone: (216) Fax: (216) E mail: kxp65@case.edu 1

2 Supplementary Figures and Legends Figure S1. Size exclusion chromatographic profile (on a Superdex 200 column) of 5 HT 4 R immuno-affinity purified from mouse rod photoreceptor cells. 2

3 Figure S2. 5 HT 4 R expressed in different systems (TRex cells, insect Sf9 cells and HEK293 cells) separated by SDS PAGE and identified by 1D4 immunoblotting. Treatment with PNGase F (plus sign at bottom) removed glycosyl residues, yielding a deglycosylated 5 HT 4 R monomer (arrows). 3

4 Figure S3. Mass spectrometry coverage of 5 HT 4 R from transgenic mice treated with trypsin (red) or chymotrypsin (blue). Sequence coverage for glycosylated 5 HT 4 R (A) and deglycosylated 5 HT 4 R (B) is shown. A 1 MDKLDANVSS EEGFGSVEKV VLLTFLSTVI LMAILGNLLV MVAVCWDRQL 51 RKIKTNYFIV SLAFADLLVS VLVMPFGAIE LVQDIWIYGE VFCLVRTSLD 101 VLLTTASIFH LCCISLDRYY AICCQPLVYR NKMTPLRIAL MLGGCWVIPT 151 FISFLPIMQG WNNIGIIDLI EKRKFNQNSN STYCVFMVNK PYAITCSVVA 201 FYIPFLLMVL AYYRIYVTAK EHAHQIQMLQ RAGASSESRP QSADQHSTHR 251 MRTETKAAKT LCIIMGCFCL CWAPFFVTNI VDPFIDYTVP GQVWTAFLWL 301 GYINSGLNPF LYAFLNKSFR RAFLIILCCD DERYRRPSIL GQTVPCSTTT 351 INGSTHVLRD AVECGGQWES QCHPPATSPL VAAQPSDT B 1 MDKLDANVSS EEGFGSVEKV VLLTFLSTVI LMAILGNLLV MVAVCWDRQL 51 RKIKTNYFIV SLAFADLLVS VLVMPFGAIE LVQDIWIYGE VFCLVRTSLD 101 VLLTTASIFH LCCISLDRYY AICCQPLVYR NKMTPLRIAL MLGGCWVIPT 151 FISFLPIMQG WNNIGIIDLI EKRKFNQNSN STYCVFMVNK PYAITCSVVA 201 FYIPFLLMVL AYYRIYVTAK EHAHQIQMLQ RAGASSESRP QSADQHSTHR 251 MRTETKAAKT LCIIMGCFCL CWAPFFVTNI VDPFIDYTVP GQVWTAFLWL 301 GYINSGLNPF LYAFLNKSFR RAFLIILCCD DERYRRPSIL GQTVPCSTTT 351 INGSTHVLRD AVECGGQWES QCHPPATSPL VAAQPSDT 4

5 Figure S4. Tandem mass spectra of two glycopeptides from 5 HT 4 R expressed in mouse rod cells. (A) Tryptic glycopeptide 4 LDANVSSEEGFGSVEK 19 with m/z of (2+). (B) Chymotryptic glycopeptide 176 NQNSNSTYCVF 186 with m/z of (3+). Collision induced dissociation of attached oligosaccharides allowed manual interpretation of the polysaccharide composition. 5

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7 Figure S5. GPCR expression in rod cells of transgenic mice yields homogenously glycosylated, well behaved receptor. The major band in each lane corresponds to cannabinoid receptor 2 (A), adenosine A1 receptor (B), and 5 HT 2C R, 5 HT 7 R (C). Purified receptors CB 2 R and A A1 R were separated by SDS PAGE and visualized after silver staining (A,B). DDM extracts of retinas from mice expressing 5 HT 2C R or 5 HT 7 R were subjected to SDS PAGE and analyzed by 1D4 immuno-blotting (C). 7

8 Figure S6. Rhodopsin expressed in two different mammalian expression systems (HEK293 cells and NIH 3T3 cells) was solubilized with DDM, separated by SDS PAGE and developed by 1D4 immunoblotting. An aliquot from each sample was treated with PNGase F (plus sign at bottom). Arrow indicates deglycosylated rhodopsin monomer. 8

9 Figure S7. Dephosphorylation of 5 HT 4 R expressed in mouse rod cells with protein phosphatase PP2A at 4 C. Three different dilutions of purified of 5 HT 4 R (lanes 1 3) and PP2A treated 5 HT 4 R (lanes 4-6) were resolved on a SDS PAGE gel, stained with the phosphoprotein stain ProQ Diamond and imaged with Typhoon Trio (GE Healthcare) with excitation at 532 nm and emission at 580 ± 30 nm (A). After fluorescent imaging, the same gel was silver stained (B). Monomer band intensities for each type of staining are plotted in panel C, with closed circles for non treated 5 HT 4 R and open circles for PP2A treated 5 HT 4 R. Numbers in the plot correspond to the lanes in panels A and B. Note that the band intensity ratio of phospho stained/silver stained is significantly lower after PP2A treatment. Residual phospho staining after PP2A treatment may be due to non-specific staining or incomplete dephosphorylation. Arrow designates the diglycosylated 5 HT 4 R monomer band. 9

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11 Figure S8. Tandem mass spectra of mouse-expressed 5 HT 4 R peptide 232 AGASSESRPQSADQHSTHR 250 suggests phosphorylation of residues S 235 (A), S 235 and S 242 (B), S 235, S 236 and S 242 (C), and S 235, S 236, S 238 and S 242 (D). The parent ions for each phosphoform are (3+), (3+), (3+) and (3+), respectively. The modified residues are showed in red. 11

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