Cross-linking of erythrocyte membrane proteins by periodate and intramembrane particle distribution.
Ontology highlight
ABSTRACT:
SUBMITTER: Gahmberg CG
PROVIDER: S-EPMC1184014 | biostudies-other | 1978 Jun
REPOSITORIES: biostudies-other
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Project description:Fundamental knowledge about how G protein-coupled receptors and their ligands interact is important for understanding receptor-ligand binding and the development of new drug discovery strategies. We have used cross-linking and tandem mass spectrometry analyses to investigate the interaction of the N terminus of the Saccharomyces cerevisiae tridecapeptide pheromone, ?-factor (WHWLQLKPGQPMY), and Ste2p, its cognate G protein-coupled receptor. The Trp(1) residue of ?-factor was replaced by 3,4-dihydroxyphenylalanine (DOPA) for periodate-mediated chemical cross-linking, and biotin was conjugated to Lys(7) for detection purposes to create the peptide [DOPA(1),Lys(7)(BioACA),Nle(12)]?-factor, called Bio-DOPA(1)-?-factor. This ligand analog was a potent agonist and bound to Ste2p with ?65 nanomolar affinity. Immunoblot analysis of purified Ste2p samples that were treated with Bio-DOPA(1)-?-factor showed that the peptide analog cross-linked efficiently to Ste2p. The cross-linking was inhibited by the presence of either native ?-factor or an ?-factor antagonist. MALDI-TOF and immunoblot analyses revealed that Bio-DOPA(1)-?-factor cross-linked to a fragment of Ste2p encompassing residues Ser(251)-Met(294). Fragmentation of the cross-linked fragment and Ste2p using tandem mass spectrometry pinpointed the cross-link point of the DOPA(1) of the ?-factor analog to the Ste2p Lys(269) side chain near the extracellular surface of the TM6-TM7 bundle. This conclusion was confirmed by a greatly diminished cross-linking of Bio-DOPA(1)-?-factor into a Ste2p(K269A) mutant. Based on these and previously obtained binding contact data, a mechanism of ?-factor binding to Ste2p is proposed. The model for bound ?-factor shows how ligand binding leads to conformational changes resulting in receptor activation of the signal transduction pathway.
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