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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Cellular Expression of the Human Angiotensin II Type 1Receptor Containing the Non-Canonical PhotolabellingAmino Acid BpaJason Arsenault, Julie Lehoux, Brian J. Holleran, Marilou Lefrançois,Gaetan Guillemette, Richard Leduc, and Emanuel EscherUniversitee de Sherbrooke, Sherbrooke, J1H 5N4, CanadaIntroductionThe incorporation of non-coded amino acids into functional proteins in mammalian cellshas recently been achieved by several laboratories, mostly through engineered tRNAaminoacyl-tRNAsynthetase pairs for the specific incorporation of unnatural amino acidsthrough recognition of an AMBER codons [1]. We applied this approach to a G ProteinCoupled Receptors (GPCR) in order to ultimately identify the binding partners (e.g.ligands, signal transduction- and regulatory proteins) of these receptors through photocrosslinking.The human angiotensin II type 1 receptor (hAT 1 ), a peptidergic GPCR, has beenextensively studied using synthetic Angiotensin II (AngII) analogues containing thephotoreactive residue p-Benzoyl-L-Phenylalanine (Bpa). This has allowed to map theligand binding domain of hAT 1 and to deduce its structure. Previous results have identifiedtransmembrane domain (TMD) 7 as interacting with position 8 of the AngII analogue125 I-[Sar 1 , Bpa 8 ] AngII [2,3]. Residues 293 and 294 were the principal contact points [2].As a proof-of-concept for later protein-receptor interaction studies we applied this approachin the aim of an inverse labeling study. The Amber codon TAG was inserted in a sitespecificmanner into the hAT 1 receptor gene at known ligand-interaction residues.Following co-transfection of this gene, together with a CUA bst tRNA, its engineeredcognate Bpa specific aminoacyl-tRNA synthetase, and in presence of free Bpa, themammalian cell line COS-7 was able to expressed hAT 1 mutants. TAG mutants wereprepared in a site-specific fashion in the proximal location to the AngII bindingenvironment. Since Bpa has been shown to react to methionine with a preferential kinetic[3], the [Sar 1 , Met 8 ] AngII analogue was synthesized. However, synthesis of nonoxidized125 I-[Sar 1 , Met 8 ] Ang II as a Bpa selective bait ligand was not successful and an alternativeidentification strategy had to be found. The constitutively active N111G hAT 1 templatetolerates N-terminal ligand modifications [4]. For this purpose, [Iminobiotinyl-Aca 0 , Gly 1 ,Met 8 ] AngII was synthesized and N111G hAT 1 was mutated with the same TAG codons toproduce the N111G/F293Bpa and N111G/C296Bpa hAT 1 . N-terminal flag and C-terminalV5 epitopes were also added for immunoblotting.Results and DiscussionAll of the F293Bpa and C296Bpa receptor mutants displayed native hAT 1 like bindingaffinity towards Ang II and [Sar 1 , Ile 8 ] Ang II albeit with reduced expression rates (1/3 to1/10 of WT expression rates). In absence of either one of the transfection elements or offree Bpa, no receptor expression was observed. As expected, all of the tested AngIIanalogues had low nM affinity towards the WT hAT 1 receptor while the N-terminaliminobiotinylated AngII analogues had nM affinity towards the N111G hAT 1 receptoronly. The hAT 1 and the Bpa mutant receptors were photolabeled at a constant 25 o C with125 I-[Sar 1 , Bpa 8 ] Ang II for 1h. Figure 1 shows that photoaffinity labeling can quicklydistinguish between complete and incomplete receptors. Receptors having a truncation atresidue 293 or 296 above the highly conserved NPxxY motif do not express at the plasmamembrane nor can they bind AngII analogues. CNBr digestion as well as western blottingconfirmed the integrity of the engineered protein. The two iminobiotinylated peptides didnot produce sufficient yields to show specific N111G/F293Bpa and N111G/C296Bpa hAT 1receptor labeling by streptavidine HRP chemiluminescence. Subsequent receptorpurification could permit the positive identification of ligand-labeled receptor complexes.However, interestingly the CNBr digestion of the 125 I-[Sar 1 , Bpa 8 ] AngII labeled C296BpahAT 1 receptor showed a slight elevation of molecular sizes in SDS-PAGE electrophoresis(see Figure 2A). According to the molecular weight ladders this would be an addition of1 to 2 kDa on the TMD 7 fragments. This thus suggested that we were tagging a smallauxiliary receptor binding protein. To confirm this finding we also submitted our receptor546