ALLOSTERIC REGULATION OF GPCR ACTIVITY BY PHOSPHOLIPIDS
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1 Supplementary Information ALLOSTERIC REGULATION OF GPCR ACTIVITY BY PHOSPHOLIPIDS Rosie Dawaliby 1, Cataldo Trubbia 1, Cédric Delporte 3,4, Matthieu Masureel 2, Pierre Van Antwerpen 3,4, Brian K. Kobilka 2* & Cédric Govaerts 1* 1 Laboratory for the Structure and Function of Biological Membranes, Center for Structural Biology and Bioinformatics, Université Libre de Bruxelles, CP 206/02, Bd du Triomphe, 1050 Brussels, Belgium 2 Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305, USA. 3 Laboratory of Pharmaceutical Chemistry Faculty of Pharmacy, Université Libre de Bruxelles, Brussels, Belgium. 4 Analytical Platform of the Faculty of Pharmacy, Faculty of Pharmacy, Université Libre de Bruxelles, Brussels, Belgium. * Correspondence and request for materials should be addressed to C.G. (Cedric.Govaerts@ulb.ac.be) and B.K.K. (kobilka@stanford.edu) 1
2 Supplementary Results Supplementary Figure 1: Chemical structure of the lipids used for in this study. For clarity the acyl chains are not shown and replaced by R1 and R2 labels. 2
3 Supplementary Figure 2: Size-exclusion of 2R rhdls of different lipidic compositions Size-exclusion chromatography profiles of the various rhdl using a Superdex 200/300GL column (GE Healthcare) showing one major peak in all cases. The main peak was collected for subsequent functional assays. 3
4 Supplementary Figure 3: Homogeneity and particle size analysis of 2R rhdls in different lipidic compositions. Transmission electron micrographs for negatively stained rhdls of different lipid compositions. Defined homogeneous particles of nm depending on the lipid used were observed. Particle sizes in each 4
5 Supplementary Figure 4: Solubilized lipids do not form bilayer Transmission electron micrographs for negatively stained samples of 2R + solubilized DOPG (1 M 2R, 200 M DOPG, 500 M DDM and 600 M sodium cholate), showing particle size of about 8 nm, as expected for receptor/detergent micelles. No liposome was observed. 5
6 Supplementary Figure 5: Modulation of 2R function by lipids does not require a bilayer. a c. Bimane fluorescence intensity was quantified by integration of the fluorescence curves areas and normalized to the no lipid curve without ligand. a. Time dependent changes in bimane fluorescence intensity of solubilized bimane-labelled 2R (1 M receptor, maintained in 500 M DDM) upon addition of saturating concentration of 200 M Isoproterenol in the presence of increasing concentration of solubilized DOPG (with 600 M Sodium Cholate). For the No lipids condition solubilized receptor was maintained in 500 M DDM and incubated with M sodium cholate without additional solubilized lipids. Data are the mean ± SEM of at 3 independent experiments. b. Time dependent changes in bimane in bimane fluorescence intensity upon addition of saturating concentration of 200 M Isoproterenol in the presence of 200 M of either DOPC, DOPG, DOPE and DOPS (detergent concentrations are identical to those in panel a). Data are the mean ± SEM of at least 3 independent experiments. c. Time dependent changes in bimane in bimane fluorescence intensity upon addition of 4 M Nb80 followed by agonist activation (200 M Isoproterenol). Data are the mean ± SEM of at least 3 independent experiments. 6
7 Supplementary table 1: 2R affinities to agonist and antagonist in different rhdl. Affinities (IC 50 ) of agonist (Isoproterenol) and antagonist (Alprenolol) for 2R reconstituted in rhdls of different lipid compositions measured by competition against [ 3 H]-dihydroalprenolol. Values represent means ± SEM of at least 3 independent experiments (see online methods). Alprenolol Isoproternol IC 50 (nm) IC 50 (µm) DOPC 8.6 ± ± 1.1 DOPE 2.8 ± ± 0.5 DOPG 9.3 ± ± 0.1 DOPS 5.9 ± ± 0.1 DOPI 8.9 ± ± 0.2 7
8 Supplementary Table 2: Lipids bound to purified 2R determined by mass spectrometry. Lipids from Sf9 membranes and Sf9 purified 2R were identified and quantified using rapid resolution liquid chromatography coupled to mass spectrometry. Relative percentage of each type of phospholipid and cholesterol is represented as mean ± SEM of 3 independent experiments. Enrichment of each lipid is calculated as the ratio of the percentage of the given lipid bound to receptor in relation to the percentage of the lipid found in Sf9 membranes. Lipids in SF9 membranes (% of total) Lipids bound to β2r (% of total) Relatie enrichement (fold) PC 15,39 33,09 2,2 PE 35,32 40,99 1,2 PG 3,65 15,91 4,4 PS 18,90 2,38 0,1 PI 13,89 4,55 0,3 SM 11,42 3,10 0,3 Cholesterol/PL 0,03 0,53 17,7 8
9 Supplementary Table 3: Specific enrichment of phosphatidylglycerol species on the 2R. Phosphatidylglycerol species found with purified 2R were identified and quantified in lipids mixes from Sf9 membranes and from lipids extracted from Sf9 purified 2R as in supplementary table 2. Relative percentage of each phosphatydilglycerol species is represented as mean ± SEM of 3 independent experiments. Enrichment of each species on the 2R relative to their specific abundance in Sf9 membranes. PG Lipids in SF9 membranes (% of total) Lipids bound to β2r (% of total) Fold enrichement on 2R C16:1 C16:1 2,25 0 C16:0 C16:1 0,23 0 C18:1 C16:1 1,01 8,70 8,6 C18:1 C18:1 0,09 7,21 80,1 C18:0 C18:1 0,07 0 9
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