Probing the topology of the glycine receptor by chemical modification coupled to mass spectrometry.
Tetranitromethane (TNM), a small aqueous reagent that specifically modifies solvent-accessible tyrosine residues to o-nitrotyrosine, was used to probe the topology of the GlyR. Homomers of human alpha1 GlyR were recombinantly expressed via a baculovirus system, affinity-purified, and reconstituted in lipid vesicles of defined composition. The native-like reconstituted receptors were then reacted with TNM, and GlyR reaction products were isolated by SDS-PAGE. After proteolytic digestion, TNM-labeled residues were identified using mass spectrometry by observing the mass shift corresponding to the nitrate moiety. In this manner, we have identified TNM modifications of tyrosine residues at positions 24, 75, 78, 161, 223, and 228 in the receptor. Of significance, nitrations at Tyr 223 and Tyr 228 occur within the first putative transmembrane helix (M1) of the receptor, and their labeling suggests a non-helical secondary structure for M1 for the glycine receptor. In a previously published report [Leite et al. (2000) J. Biol. Chem. 275, 13683], we also identified proteolytic cleavage sites within M1. Taken together, these studies support a topological model where the "historical" M1 segment cannot be entirely alpha-helical and may contain an extramembranous surface loop. Furthermore, we have also identified a tyrosine modification (Tyr 161) within a region of the N-terminal domain critical in agonist and antagonist binding.
Leite
John F
JF
Department of Molecular Genetics and Biochemistry, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
Cascio
Michael
M
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D006801:Humans; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D018009:Receptors, Glycine / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013774:Tetranitromethane; D014443:Tyrosine / Q000737:chemistry
D018009:Receptors, Glycine; D011994:Recombinant Proteins; D014443:Tyrosine; D013774:Tetranitromethane